Insecticidal proteins and methods for their use

ABSTRACT

Compositions and methods for controlling pests are provided. The methods involve transforming organisms with a nucleic acid sequence encoding an insecticidal protein. In particular, the nucleic acid sequences are useful for preparing plants and microorganisms that possess insecticidal activity. Thus, transformed bacteria, plants, plant cells, plant tissues and seeds are provided. Compositions are insecticidal nucleic acids and proteins of bacterial species. The sequences find use in the construction of expression vectors for subsequent transformation into organisms of interest including plants, as probes for the isolation of other homologous (or partially homologous) genes. The pesticidal proteins find use in controlling, inhibiting growth or killing Lepidopteran, Coleopteran, Dipteran, fungal, Hemipteran and nematode pest populations and for producing compositions with insecticidal activity.

CROSS REFERENCE

This application is a 371 (National Stage) of PCT/US14/55128 filed Sep.11, 2014, which claims the benefit of U.S. Provisional Application No.62/877,625 filed Sep. 13, 2013, which are incorporated herein byreference in their entirety.

A sequence listing having the file name “5345PCT_sequence_listing.txt”created on Aug. 28, 2014, and having a size of 576 kilobytes is filed incomputer readable form concurrently with the specification. The sequencelisting is part of the specification and is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

This disclosure relates to the field of molecular biology. Provided arenovel genes that encode pesticidal proteins. These pesticidal proteinsand the nucleic acid sequences that encode them are useful in preparingpesticidal formulations and in the production of transgenicpest-resistant plants.

BACKGROUND OF THE INVENTION

Biological control of insect pests of agricultural significance using amicrobial agent, such as fungi, bacteria or another species of insectaffords an environmentally friendly and commercially attractivealternative to synthetic chemical pesticides. Generally speaking, theuse of biopesticides presents a lower risk of pollution andenvironmental hazards and biopesticides provide greater targetspecificity than is characteristic of traditional broad-spectrumchemical insecticides. In addition, biopesticides often cost less toproduce and thus improve economic yield for a wide variety of crops.

Certain species of microorganisms of the genus Bacillus are known topossess pesticidal activity against a range of insect pests includingLepidoptera, Diptera, Coleoptera, Hemiptera and others. Bacillusthuringiensis (Bt) and Bacillus popilliae are among the most successfulbiocontrol agents discovered to date. Insect pathogenicity has also beenattributed to strains of B. larvae, B. lentimorbus, B. sphaericus and B.cereus. Microbial insecticides, particularly those obtained fromBacillus strains, have played an important role in agriculture asalternatives to chemical pest control.

Crop plants have been developed with enhanced insect resistance bygenetically engineering crop plants to produce pesticidal proteins fromBacillus. For example, corn and cotton plants have been geneticallyengineered to produce pesticidal proteins isolated from strains of Bt.These genetically engineered crops are now widely used in agricultureand have provided the farmer with an environmentally friendlyalternative to traditional insect-control methods. While they haveproven to be very successful commercially, these genetically engineered,insect-resistant crop plants provide resistance to only a narrow rangeof the economically important insect pests. In some cases, insects candevelop resistance to different insecticidal compounds, which raises theneed to identify alternative biological control agents for pest control.

Accordingly, there remains a need for new pesticidal proteins withdifferent ranges of insecticidal activity against insect pests, e.g.,insecticidal proteins which are active against a variety of insects inthe order Lepidoptera and the order Coleoptera including but not limitedto insect pests that have developed resistance to existing insecticides.

SUMMARY OF THE INVENTION

Compositions and methods for conferring pesticidal activity to bacteria,plants, plant cells, tissues and seeds are provided. Compositionsinclude nucleic acid molecules encoding sequences for pesticidal andinsecticidal polypeptides, vectors comprising those nucleic acidmolecules, and host cells comprising the vectors. Compositions alsoinclude the pesticidal polypeptide sequences and antibodies to thosepolypeptides. The nucleic acid sequences can be used in DNA constructsor expression cassettes for transformation and expression in organisms,including microorganisms and plants. The nucleotide or amino acidsequences may be synthetic sequences that have been designed forexpression in an organism including, but not limited to, a microorganismor a plant. Compositions also comprise transformed bacteria, plants,plant cells, tissues and seeds.

In particular, isolated or recombinant nucleic acid molecules areprovided encoding Pseudomonas Insecticidal Protein-72 (PIP-72)polypeptides including amino acid substitutions, deletions, insertions,and fragments thereof, and combinations thereof. Additionally, aminoacid sequences corresponding to the PIP-72 polypeptides are encompassed.Provided are isolated or recombinant nucleic acid molecules capable ofencoding a PIP-72 polypeptide of SEQ ID NO: 849 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof. Nucleic acid sequences that are complementary to a nucleic acidsequence of the embodiments or that hybridize to a sequence of theembodiments are also encompassed. Also provided are isolated orrecombinant PIP-72 polypeptides of SEQ ID NO: 849 as well as amino acidsubstitutions, deletions, insertions, fragments thereof and combinationsthereof.

Methods are provided for producing the polypeptides and for using thosepolypeptides for controlling or killing a Lepidopteran, Coleopteran,nematode, fungi, and/or Dipteran pests. The transgenic plants of theembodiments express one or more of the pesticidal sequences disclosedherein. In various embodiments, the transgenic plant further comprisesone or more additional genes for insect resistance, for example, one ormore additional genes for controlling Coleopteran, Lepidopteran,Hemipteran or nematode pests. It will be understood by one of skill inthe art that the transgenic plant may comprise any gene imparting anagronomic trait of interest.

Methods for detecting the nucleic acids and polypeptides of theembodiments in a sample are also included. A kit for detecting thepresence of a PIP-72 polypeptide or detecting the presence of anucleotide sequence encoding a PIP-72 polypeptide in a sample isprovided. The kit may be provided along with all reagents and controlsamples necessary for carrying out a method for detecting the intendedagent, as well as instructions for use.

The compositions and methods of the embodiments are useful for theproduction of organisms with enhanced pest resistance or tolerance.These organisms and compositions comprising the organisms are desirablefor agricultural purposes. The compositions of the embodiments are alsouseful for generating altered or improved proteins that have pesticidalactivity or for detecting the presence of PIP-72 polypeptides or nucleicacids in products or organisms.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 shows the amino acid sequence alignment of PIP-72Aa (SEQ ID NO:2), PIP-72Ba (SEQ ID NO: 4); PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ IDNO: 8); PIP-72 Da (SEQ ID NO: 10); PIP-72Db (SEQ ID NO: 12); PIP-72Dc(SEQ ID NO: 14); PIP-72Ea (SEQ ID NO: 16), PIP-72Fa (SEQ ID NO: 18);GBP_A3175 (SEQ ID NO: 20), SRBS_294080 (SEQ ID NO: 22); JG43047 (SEQ IDNO: 24); SwiRh_4910 (SEQ ID NO: 26); PIP-72Ff (SEQ ID NO: 28), PFL_6283(SEQ ID NO: 30); PIP-72Gb (SEQ ID NO: 32); XBJ1_1078 (SEQ ID NO: 34);plu2373 (SEQ ID NO: 36); and PIP-72Ge (SEQ ID NO: 38). The sequencediversity is highlighted. Amino acids 37-51 (Motif 1) relative toPIP-72Aa (SEQ ID NO: 2) are underlined.

FIG. 2 shows an alignment of the amino acid sequences of PIP-72Aa (SEQID NO: 2), PIP-72Ab (SEQ ID NO: 927); PIP-72Ba (SEQ ID NO: 4); PIP-72Bb(SEQ ID NO: 928); PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ ID NO: 8);WP_030131237 (SEQ ID NO: 929); PIP-72 Da (SEQ ID NO: 10); PIP-72Db (SEQID NO: 12); PIP-72Dc (SEQ ID NO: 14); PIP-72Fa (SEQ ID NO: 18); andGBP_A3175 (SEQ ID NO: 20). The amino acid diversity between PIP-72Aa(SEQ ID NO: 2) and the other homologs is indicated with shading.

FIG. 3 shows the amino acid sequence alignment of PIP-72Aa (SEQ ID NO:2), PIP-72Ba (SEQ ID NO: 4); PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ IDNO: 8); PIP-72 Da (SEQ ID NO: 10); PIP-72Db (SEQ ID NO: 12); andPIP-72Dc (SEQ ID NO: 14). The amino acid diversity between PIP-72Aa (SEQID NO: 2) and the other homologs is indicated with shading.

FIG. 4 shows the amino acid sequence alignment of WP_030131237 (SEQ IDNO: 929) PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ ID NO: 8); PIP-72 Da(SEQ ID NO: 10); PIP-72Db (SEQ ID NO: 12); and PIP-72Dc (SEQ ID NO: 14).The amino acid diversity between PIP-72 Da (SEQ ID NO: 10) and the otherhomologs is indicated with shading.

FIG. 5 shows an alignment of the amino acid sequences of PIP-72Fh (SEQID NO: 932), PIP-72Gi (SEQ ID NO: 941); PIP-72Fi (SEQ ID NO: 933);PIP-72GI (SEQ ID NO: 944); PIP-72Fa (SEQ ID NO: 14). The amino aciddiversity between PIP-72Ca (SEQ ID NO: 2) and the other homologs isindicated with shading.

FIG. 6 shows the T0 GH efficacy results for events generated from thePHP61664, PHP61666, PHP61668, PHP64465, PHP64468, PHP64471, and PHP69828constructs. Efficacy for events derived from the constructs was observedrelative to negative control events as measured by root protection fromWestern corn rootworm. Root protection was measured according to thenumber of nodes of roots injured (CRWNIS=corn rootworm node injuryscore) using the method developed by Oleson, et al. (2005) [J. EconEntomol. 98(1):1-8]. The root injury score is measured from “0” to “3”with “0” indicating no visible root injury, “1” indicating 1 node ofroot damage, “2” indicating 2 nodes or root damage, and “3” indicating amaximum score of 3 nodes of root damage. Each symbol (triangle, squareor circle) represents a single event.

DETAILED DESCRIPTION

It is to be understood that this disclosure is not limited to theparticular methodology, protocols, cell lines, genera, and reagentsdescribed, as such may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentdisclosure.

As used herein the singular forms “a”, “and”, and “the” include pluralreferents unless the context clearly dictates otherwise. Thus, forexample, reference to “a cell” includes a plurality of such cells andreference to “the protein” includes reference to one or more proteinsand equivalents thereof known to those skilled in the art, and so forth.All technical and scientific terms used herein have the same meaning ascommonly understood to one of ordinary skill in the art to which thisdisclosure belongs unless clearly indicated otherwise.

The present disclosure is drawn to compositions and methods forcontrolling pests. The methods involve transforming organisms withnucleic acid sequences encoding a PIP-72 polypeptide. In particular, thenucleic acid sequences of the embodiments are useful for preparingplants and microorganisms that possess pesticidal activity. Thus,transformed bacteria, plants, plant cells, plant tissues and seeds areprovided. The compositions are pesticidal nucleic acids and proteins ofbacterial species. The nucleic acid sequences find use in theconstruction of expression vectors for subsequent transformation intoorganisms of interest, as probes for the isolation of other homologous(or partially homologous) genes, and for the generation of alteredPIP-72 polypeptides by methods known in the art, such as site-directedmutagenesis, domain swapping or DNA shuffling. The PIP-72 polypeptidesfind use in controlling or killing Lepidopteran, Coleopteran, Dipteran,fungal, Hemipteran and nematode pest populations and for producingcompositions with pesticidal activity. Insect pests of interest include,but are not limited to, Lepidoptera species including but not limitedto: diamond-back moth, e.g., Helicoverpa zea Boddie; soybean looper,e.g., Pseudoplusia includens Walker; and velvet bean caterpillar e.g.,Anticarsia gemmatalis Hübner and Coleoptera species including but notlimited to Western corn rootworm (Diabrotica virgifera)—WCRW, Southerncorn rootworm (Diabrotica undecimpunctata howardi)—SCRW, and Northerncorn rootworm (Diabrotica barberi)—NCRW.

By “pesticidal toxin” or “pesticidal protein” is used herein to refer toa toxin that has toxic activity against one or more pests, including,but not limited to, members of the Lepidoptera, Diptera, Hemiptera andColeoptera orders or the Nematoda phylum or a protein that has homologyto such a protein. Pesticidal proteins have been isolated from organismsincluding, for example, Bacillus sp., Pseudomonas sp., Photorhabdus sp.,Xenorhabdus sp., Clostridium bifermentans and Paenibacillus popilliae.Pesticidal proteins include but are not limited to: insecticidalproteins from Pseudomonas sp. such as PSEEN3174 (Monalysin; (2011) PLoSPathogens 7:1-13); from Pseudomonas protegens strain CHA0 and Pf-5(previously fluorescens) (Pechy-Tarr, (2008) Environmental Microbiology10:2368-2386; GenBank Accession No. EU400157); from PseudomonasTaiwanensis (Liu, et al., (2010) J. Agric. Food Chem., 58:12343-12349)and from Pseudomonas pseudoalcligenes (Zhang, et al., (2009) Annals ofMicrobiology 59:45-50 and Li, et al., (2007) Plant Cell Tiss. OrganCult. 89:159-168); insecticidal proteins from Photorhabdus sp. andXenorhabdus sp. (Hinchliffe, et al., (2010) The Open Toxicology Journal,3:101-118 and Morgan, et al., (2001) Applied and Envir. Micro.67:2062-2069); U.S. Pat. Nos. 6,048,838, and 6,379,946; a PIP-1polypeptide of U.S. Ser. No. 13/792,861; an AfIP-1A and/or AfIP-1Bpolypeptides of U.S. Ser. No. 13/800,233; a PHI-4 polypeptides of U.S.Ser. No. 13/839,702; PIP-47 polypeptides of U.S. Ser. No. 61/866,747;the insecticidal proteins of U.S. Ser. Nos. 61/863,761 and 61/863763;and δ-endotoxins including but not limited to: the Cry1, Cry2, Cry3,Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14,Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24,Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34,Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44,Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53, Cry 54, Cry55,Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63, Cry64, Cry65,Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 classes of δ-endotoxin genesand the B. thuringiensis cytolytic cyt1 and cyt2 genes. Members of theseclasses of B. thuringiensis insecticidal proteins include, but are notlimited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession #Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4(Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6(Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8(Accession #I26149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession# AAD55382); Cry1Aa11 (Accession # CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19 (Accession # HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession # AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession # CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #I12419); Cry1Ab12 (Accession # AAC64003);Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877);Cry1Ab15 (Accession # AAO13302); Cry1Ab16 (Accession # AAK55546);Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259);Cry1Ab19 (Accession # AAW31761); Cry1Ab20 (Accession # ABB72460);Cry1Ab21 (Accession # ABS18384); Cry1Ab22 (Accession # ABW87320);Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778);Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729);Cry1Ab27 (Accession # JN135249); Cry1Ab28 (Accession # JN135250);Cry1Ab29 (Accession # JN135251); Cry1Ab30 (Accession # JN135252);Cry1Ab31 (Accession # JN135253); Cry1Ab32 (Accession # JN135254);Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668);Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337);Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858);Cry1Ac1 (Accession # AAA22331); Cry1Ac2 (Accession # AAA22338); Cry1Ac3(Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5(Accession # AAA22339); Cry1Ac6 (Accession # AAA86266); Cry1Ac7(Accession # AAB46989); Cry1Ac8 (Accession # AAC44841); Cry1Ac9(Accession # AAB49768); Cry1Ac10 (Accession # CAA05505); Cry1Ac11(Accession # CAA10270); Cry1Ac12 (Accession #I12418); Cry1Ac13(Accession # AAD38701); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15(Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17(Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19(Accession # ABD37053); Cry1Ac20 (Accession # ABB89046); Cry1Ac21(Accession # AAY66992); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23(Accession # CAQ30431); Cry1Ac24 (Accession # ABL01535); Cry1Ac25(Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27(Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29(Accession # DQ438941); Cry1Ac30 (Accession # GQ227507); Cry1Ac31(Accession # GU446674); Cry1Ac32 (Accession # HM061081); Cry1Ac33(Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35(Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37(Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2(Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1(Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1(Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3(Accession # HQ439779); Cry1Ai1 (Accession # AAO39719); Cry1Ai2(Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1(Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3(Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5(Accession # AB020894); Cry1Ba6 (Accession # ABL60921); Cry1Ba7(Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2(Accession # HQ439782); Cry1Bc1 (Accession # CAA86568); Cry1Bd1(Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1(Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3(Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1(Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1(Accession # AAO39720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1(Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2(Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4(Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6 [1](Accession # AAF37224); Cry1Ca7 (Accession # AAG50438); Cry1Ca8(Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10(Accession # AAN16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12(Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14(Accession # JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession# AAG35409); Cry1Cb3 (Accession # ACD50894); Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession #I76415);Cry1Da3 (Accession # HQ439784); Cry1 Db1 (Accession # CAA80234); Cry1Db2 (Accession # AAK48937); Cry1 Dc1 (Accession # ABK35074); Cry1Ea1(Accession # CAA37933); Cry1Ea2 (Accession # CAA39609); Cry1Ea3(Accession # AAA22345); Cry1Ea4 (Accession # AAD04732); Cry1Ea5(Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession# AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession #HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession #JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession #AAA22348); Cry1Fa2 (Accession # AAA22347); Cry1Fa3 (Accession #HM070028); Cry1Fa4 (Accession # HM439638); Cry1 Fb1 (Accession #CAA80235); Cry1Fb2 (Accession # BAA25298); Cry1Fb3 (Accession #AAF21767); Cry1Fb4 (Accession # AAC10641); Cry1Fb5 (Accession #AAO13295); Cry1Fb6 (Accession # ACD50892); Cry1Fb7 (Accession #ACD50893); Cry1Ga1 (Accession # CAA80233); Cry1Ga2 (Accession #CAA70506); Cry1Gb1 (Accession # AAD10291); Cry1Gb2 (Accession #AAO13756); Cry1Gc1 (Accession # AAQ52381); Cry1Ha1 (Accession #CAA80236); Cry1Hb1 (Accession # AAA79694); Cry1Hb2 (Accession #HQ439786); Cry1H-like (Accession # AAF01213); Cry1Ia1 (Accession #CAA44633); Cry1Ia2 (Accession # AAA22354); Cry1Ia3 (Accession #AAC36999); Cry1Ia4 (Accession # AAB00958); Cry1Ia5 (Accession #CAA70124); Cry1Ia6 (Accession # AAC26910); Cry1Ia7 (Accession #AAM73516); Cry1Ia8 (Accession # AAK66742); Cry1Ia9 (Accession #AAQ08616); Cry1Ia10 (Accession # AAP86782); Cry1Ia11 (Accession #CAC85964); Cry1Ia12 (Accession # AAV53390); Cry1Ia13 (Accession #ABF83202); Cry1Ia14 (Accession # ACG63871); Cry1Ia15 (Accession #FJ617445); Cry1Ia16 (Accession # FJ617448); Cry1Ia17 (Accession #GU989199); Cry1Ia18 (Accession # ADK23801); Cry1Ia19 (Accession #HQ439787); Cry1Ia20 (Accession # JQ228426); Cry1Ia21 (Accession #JQ228424); Cry1Ia22 (Accession # JQ228427); Cry1Ia23 (Accession #JQ228428); Cry1Ia24 (Accession # JQ228429); Cry1Ia25 (Accession #JQ228430); Cry1Ia26 (Accession # JQ228431); Cry1Ia27 (Accession #JQ228432); Cry1Ia28 (Accession # JQ228433); Cry1Ia29 (Accession #JQ228434); Cry1Ia30 (Accession # JQ317686); Cry1Ia31 (Accession #JX944038); Cry1Ia32 (Accession # JX944039); Cry1Ia33 (Accession #JX944040); Cry1Ib1 (Accession # AAA82114); Cry1Ib2 (Accession #ABW88019); Cry1Ib3 (Accession # ACD75515); Cry1Ib4 (Accession #HM051227); Cry1Ib5 (Accession # HM070028); Cry1Ib6 (Accession #ADK38579); Cry1Ib7 (Accession # JN571740); Cry1Ib18 (Accession #JN675714); Cry1Ib9 (Accession # JN675715); Cry1Ib10 (Accession #JN675716); Cry1Ib11 (Accession # JQ228423); Cry1Ic1 (Accession #AAC62933); Cry1Ic2 (Accession # AAE71691); Cry1Id1 (Accession #AAD44366); Cry1Id2 (Accession # JQ228422); Cry1Ie1 (Accession #AAG43526); Cry1Ie2 (Accession # HM439636); Cry1Ie3 (Accession #KC156647); Cry1Ie4 (Accession # KC156681); Cry1If1 (Accession #AAQ52382); Cry1Ig1 (Accession # KC156701); Cry1I-like (Accession #AAC31094); Cry1I-like (Accession # ABG88859); Cry1Ja1 (Accession #AAA22341); Cry1Ja2 (Accession # HM070030); Cry1Ja3 (Accession #JQ228425); Cry1Jb1 (Accession # AAA98959); Cry1Jc1 (Accession #AAC31092); Cry1Jc2 (Accession # AAQ52372); Cry1Jd1 (Accession #CAC50779); Cry1Ka1 (Accession # AAB00376); Cry1Ka2 (Accession #HQ439783); Cry1La1 (Accession # AAS60191); Cry1La2 (Accession #HM070031); Cry1Ma1 (Accession # FJ884067); Cry1Ma2 (Accession #KC156659); Cry1Na1 (Accession # KC156648); Cry1Nb1 (Accession #KC156678); Cry1-like (Accession # AAC31091); Cry2Aa1 (Accession #AAA22335); Cry2Aa2 (Accession # AAA83516); Cry2Aa3 (Accession # D86064);Cry2Aa4 (Accession # AAC04867); Cry2Aa5 (Accession # CAA10671); Cry2Aa6(Accession # CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8(Accession # AAO13734); Cry2Aa9 (Accession # AAO13750); Cry2Aa10(Accession # AAQ04263); Cry2Aa11 (Accession # AAQ52384); Cry2Aa12(Accession # AB183671); Cry2Aa13 (Accession # ABL01536); Cry2Aa14(Accession # ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1(Accession # AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3(Accession # AAG36762); Cry2Ab4 (Accession # AAO13296); Cry2Ab5(Accession # AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7(Accession # AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9(Accession # ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab11(Accession # CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13(Accession # ACG76120); Cry2Ab14 (Accession # ACG76121); Cry2Ab15(Accession # HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17(Accession # HQ439789); Cry2Ab18 (Accession # JN135255); Cry2Ab19(Accession # JN135256); Cry2Ab20 (Accession # JN135257); Cry2Ab21(Accession # JN135258); Cry2Ab22 (Accession # JN135259); Cry2Ab23(Accession # JN135260); Cry2Ab24 (Accession # JN135261); Cry2Ab25(Accession # JN415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27(Accession # JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1(Accession # CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3(Accession # AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5(Accession # ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7(Accession # CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9(Accession # CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac11(Accession # CAM83895); Cry2Ac12 (Accession # CAM83896); Cry2Ad1(Accession # AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3(Accession # CAK29504); Cry2Ad4 (Accession # CAM32331); Cry2Ad5(Accession # CAO78739); Cry2Ae1 (Accession # AAQ52362); Cry2Af1(Accession # AB030519); Cry2Af2 (Accession # GQ866915); Cry2Ag1(Accession # ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2(Accession # ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4(Accession # KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj(Accession #); Cry2Ak1 (Accession # KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession # AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession # CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession # AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession # CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession # AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession # AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession # CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession # JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession # AAA74198); Cry3Bb3 (Accession #I15475);Cry3Ca1 (Accession # CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2(Accession # BAA00179); Cry4Aa3 (Accession # CAD30148); Cry4Aa4(Accession # AFB18317); Cry4A-like (Accession # AAY96321); Cry4Ba1(Accession # CAA30312); Cry4Ba2 (Accession # CAA30114); Cry4Ba3(Accession # AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5(Accession # CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1(Accession # EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2(Accession # FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1(Accession # AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1(Accession #I34543); Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession# AAA68598); Cry5Ba2 (Accession # ABW88931); Cry5Ba3 (Accession #AFJ04417); Cry5Ca1 (Accession # HM461869); Cry5Ca2 (Accession #ZP_04123426); Cry5Da1 (Accession # HM461870); Cry5Da2 (Accession #ZP_04123980); Cry5Ea1 (Accession # HM485580); Cry5Ea2 (Accession #ZP_04124038); Cry6Aa1 (Accession # AAA22357); Cry6Aa2 (Accession #AAM46849); Cry6Aa3 (Accession # ABH03377); Cry6Ba1 (Accession #AAA22358); Cry7Aa1 (Accession # AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2 (Accession # AAA21121); Cry7Ab3 (Accession #ABX24522); Cry7Ab4 (Accession # EU380678); Cry7Ab5 (Accession #ABX79555); Cry7Ab6 (Accession # ACI44005); Cry7Ab7 (Accession #ADB89216); Cry7Ab8 (Accession # GU145299); Cry7Ab9 (Accession #ADD92572); Cry7Ba1 (Accession # ABB70817); Cry7Bb1 (Accession #KC156653); Cry7Ca1 (Accession # ABR67863); Cry7Cb1 (Accession #KC156698); Cry7Da1 (Accession # ACQ99547); Cry7Da2 (Accession #HM572236); Cry7Da3 (Accession # KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2 (Accession # HM132124); Cry7Ea3 (Accession #EEM19403); Cry7Fa1 (Accession # HM035088); Cry7Fa2 (Accession #EEM19090); Cry7Fb1 (Accession # HM572235); Cry7Fb2 (Accession #KC156682); Cry7Ga1 (Accession # HM572237); Cry7Ga2 (Accession #KC156669); Cry7Gb1 (Accession # KC156650); Cry7Gc1 (Accession #KC156654); Cry7Gd1 (Accession # KC156697); Cry7Ha1 (Accession #KC156651); Cry7Ia1 (Accession # KC156665); Cry7Ja1 (Accession #KC156671); Cry7Ka1 (Accession # KC156680); Cry7Kb1 (Accession #BAM99306); Cry7La1 (Accession # BAM99307); Cry8Aa1 (Accession #AAA21117); Cry8Ab1 (Accession # EU044830); Cry8Ac1 (Accession #KC156662); Cry8Ad1 (Accession # KC156684); Cry8Ba1 (Accession #AAA21118); Cry8Bb1 (Accession # CAD57542); Cry8Bc1 (Accession #CAD57543); Cry8Ca1 (Accession # AAA21119); Cry8Ca2 (Accession #AAR98783); Cry8Ca3 (Accession # EU625349); Cry8Ca4 (Accession #ADB54826); Cry8Da1 (Accession # BAC07226); Cry8Da2 (Accession #BD133574); Cry8Da3 (Accession # BD133575); Cry8Db1 (Accession #BAF93483); Cry8Ea1 (Accession # AAQ73470); Cry8Ea2 (Accession #EU047597); Cry8Ea3 (Accession # KC855216); Cry8Fa1 (Accession #AAT48690); Cry8Fa2 (Accession # HQ174208); Cry8Fa3 (Accession #AFH78109); Cry8Ga1 (Accession # AAT46073); Cry8Ga2 (Accession #ABC42043); Cry8Ga3 (Accession # FJ198072); Cry8Ha1 (Accession #AAW81032); Cry8Ia1 (Accession # EU381044); Cry8Ia2 (Accession #GU073381); Cry8Ia3 (Accession # HM044664); Cry8Ia4 (Accession #KC156674); Cry8Ib1 (Accession # GU325772); Cry8Ib2 (Accession #KC156677); Cry8Ja1 (Accession # EU625348); Cry8Ka1 (Accession #FJ422558); Cry8Ka2 (Accession # ACN87262); Cry8Kb1 (Accession #HM123758); Cry8Kb2 (Accession # KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1 (Accession # HM044665); Cry8Ma2 (Accession #EEM86551); Cry8Ma3 (Accession # HM210574); Cry8Na1 (Accession #HM640939); Cry8Pa1 (Accession # HQ388415); Cry8Qa1 (Accession #HQ441166); Cry8Qa2 (Accession # KC152468); Cry8Ra1 (Accession #AFP87548); Cry8Sa1 (Accession # JQ740599); Cry8Ta1 (Accession #KC156673); Cry8-like (Accession # FJ770571); Cry8-like (Accession #ABS53003); Cry9Aa1 (Accession # CAA41122); Cry9Aa2 (Accession #CAA41425); Cry9Aa3 (Accession # GQ249293); Cry9Aa4 (Accession #GQ249294); Cry9Aa5 (Accession # JX174110); Cry9Aa like (Accession #AAQ52376); Cry9Ba1 (Accession # CAA52927); Cry9Ba2 (Accession #GU299522); Cry9Bb1 (Accession # AAV28716); Cry9Ca1 (Accession #CAA85764); Cry9Ca2 (Accession # AAQ52375); Cry9Da1 (Accession #BAA19948); Cry9Da2 (Accession # AAB97923); Cry9Da3 (Accession #GQ249293); Cry9Da4 (Accession # GQ249297); Cry9Db1 (Accession #AAX78439); Cry9Dc1 (Accession # KC156683); Cry9Ea1 (Accession #BAA34908); Cry9Ea2 (Accession # AAO12908); Cry9Ea3 (Accession #ABM21765); Cry9Ea4 (Accession # ACE88267); Cry9Ea5 (Accession #ACF04743); Cry9Ea6 (Accession # ACG63872); Cry9Ea7 (Accession #FJ380927); Cry9Ea8 (Accession # GQ249292); Cry9Ea9 (Accession #JN651495); Cry9Eb1 (Accession # CAC50780); Cry9Eb2 (Accession #GQ249298); Cry9Eb3 (Accession # KC156646); Cry9Ec1 (Accession #AAC63366); Cry9Ed1 (Accession # AAX78440); Cry9Ee1 (Accession #GQ249296); Cry9Ee2 (Accession # KC156664); Cry9Fa1 (Accession #KC156692); Cry9Ga1 (Accession # KC156699); Cry9-like (Accession #AAC63366); Cry10Aa1 (Accession # AAA22614); Cry10Aa2 (Accession #E00614); Cry10Aa3 (Accession # CAD30098); Cry10Aa4 (Accession #AFB18318); Cry10A-like (Accession # DQ167578); Cry11Aa1 (Accession #AAA22352); Cry11Aa2 (Accession # AAA22611); Cry11Aa3 (Accession #CAD30081); Cry11Aa4 (Accession # AFB18319); Cry11Aa-like (Accession #DQ166531); Cry11Ba1 (Accession # CAA60504); Cry11Bb1 (Accession #AAC97162); Cry11Bb2 (Accession # HM068615); Cry12Aa1 (Accession #AAA22355); Cry13Aa1 (Accession # AAA22356); Cry14Aa1 (Accession #AAA21516); Cry14Ab1 (Accession # KC156652); Cry15Aa1 (Accession #AAA22333); Cry16Aa1 (Accession # CAA63860); Cry17Aa1 (Accession #CAA67841); Cry18Aa1 (Accession # CAA67506); Cry18Ba1 (Accession #AAF89667); Cry18Ca1 (Accession # AAF89668); Cry19Aa1 (Accession #CAA68875); Cry19Ba1 (Accession # BAA32397); Cry19Ca1 (Accession #AFM37572); Cry20Aa1 (Accession # AAB93476); Cry20Ba1 (Accession #ACS93601); Cry20Ba2 (Accession # KC156694); Cry20-like (Accession #GQ144333); Cry21Aa1 (Accession #I32932); Cry21Aa2 (Accession #I66477);Cry21Ba1 (Accession # BAC06484); Cry21Ca1 (Accession # JF521577);Cry21Ca2 (Accession # KC156687); Cry21Da1 (Accession # JF521578);Cry22Aa1 (Accession #I34547); Cry22Aa2 (Accession # CAD43579); Cry22Aa3(Accession # ACD93211); Cry22Ab1 (Accession # AAK50456); Cry22Ab2(Accession # CAD43577); Cry22Ba1 (Accession # CAD43578); Cry22Bb1(Accession # KC156672); Cry23Aa1 (Accession # AAF76375); Cry24Aa1(Accession # AAC61891); Cry24Ba1 (Accession # BAD32657); Cry24Ca1(Accession # CAJ43600); Cry25Aa1 (Accession # AAC61892); Cry26Aa1(Accession # AAD25075); Cry27Aa1 (Accession # BAA82796); Cry28Aa1(Accession # AAD24189); Cry28Aa2 (Accession # AAG00235); Cry29Aa1(Accession # CAC80985); Cry30Aa1 (Accession # CAC80986); Cry30Ba1(Accession # BAD00052); Cry30Ca1 (Accession # BAD67157); Cry30Ca2(Accession # ACU24781); Cry30Da1 (Accession # EF095955); Cry30Db1(Accession # BAE80088); Cry30Ea1 (Accession # ACC95445); Cry30Ea2(Accession # FJ499389); Cry30Fa1 (Accession # ACI22625); Cry30Ga1(Accession # ACG60020); Cry30Ga2 (Accession # HQ638217); Cry31Aa1(Accession # BAB11757); Cry31Aa2 (Accession # AAL87458); Cry31Aa3(Accession # BAE79808); Cry31Aa4 (Accession # BAF32571); Cry31Aa5(Accession # BAF32572); Cry31Aa6 (Accession # BA144026); Cry31Ab1(Accession # BAE79809); Cry31Ab2 (Accession # BAF32570); Cry31Ac1(Accession # BAF34368); Cry31Ac2 (Accession # AB731600); Cry31Ad1(Accession # BA144022); Cry32Aa1 (Accession # AAG36711); Cry32Aa2(Accession # GU063849); Cry32Ab1 (Accession # GU063850); Cry32Ba1(Accession # BAB78601); Cry32Ca1 (Accession # BAB78602); Cry32Cb1(Accession # KC156708); Cry32Da1 (Accession # BAB78603); Cry32Ea1(Accession # GU324274); Cry32Ea2 (Accession # KC156686); Cry32Eb1(Accession # KC156663); Cry32Fa1 (Accession # KC156656); Cry32Ga1(Accession # KC156657); Cry32Ha1 (Accession # KC156661); Cry32Hb1(Accession # KC156666); Cry32Ia1 (Accession # KC156667); Cry32Ja1(Accession # KC156685); Cry32Ka1 (Accession # KC156688); Cry32La1(Accession # KC156689); Cry32Ma1 (Accession # KC156690); Cry32Mb1(Accession # KC156704); Cry32Na1 (Accession # KC156691); Cry32Oa1(Accession # KC156703); Cry32Pa1 (Accession # KC156705); Cry32Qa1(Accession # KC156706); Cry32Ra1 (Accession # KC156707); Cry32Sa1(Accession # KC156709); Cry32Ta1 (Accession # KC156710); Cry32Ua1(Accession # KC156655); Cry33Aa1 (Accession # AAL26871); Cry34Aa1(Accession # AAG50341); Cry34Aa2 (Accession # AAK64560); Cry34Aa3(Accession # AAT29032); Cry34Aa4 (Accession # AAT29030); Cry34Ab1(Accession # AAG41671); Cry34Ac1 (Accession # AAG50118); Cry34Ac2(Accession # AAK64562); Cry34Ac3 (Accession # AAT29029); Cry34Ba1(Accession # AAK64565); Cry34Ba2 (Accession # AAT29033); Cry34Ba3(Accession # AAT29031); Cry35Aa1 (Accession # AAG50342); Cry35Aa2(Accession # AAK64561); Cry35Aa3 (Accession # AAT29028); Cry35Aa4(Accession # AAT29025); Cry35Ab1 (Accession # AAG41672); Cry35Ab2(Accession # AAK64563); Cry35Ab3 (Accession # AY536891); Cry35Ac1(Accession # AAG50117); Cry35Ba1 (Accession # AAK64566); Cry35Ba2(Accession # AAT29027); Cry35Ba3 (Accession # AAT29026); Cry36Aa1(Accession # AAK64558); Cry37Aa1 (Accession # AAF76376); Cry38Aa1(Accession # AAK64559); Cry39Aa1 (Accession # BAB72016); Cry40Aa1(Accession # BAB72018); Cry40Ba1 (Accession # BAC77648); Cry40Ca1(Accession # EU381045); Cry40Da1 (Accession # ACF15199); Cry41Aa1(Accession # BAD35157); Cry41Ab1 (Accession # BAD35163); Cry41Ba1(Accession # HM461871); Cry41Ba2 (Accession # ZP_04099652); Cry42Aa1(Accession # BAD35166); Cry43Aa1 (Accession # BAD15301); Cry43Aa2(Accession # BAD95474); Cry43Ba1 (Accession # BAD15303); Cry43Ca1(Accession # KC156676); Cry43Cb1 (Accession # KC156695); Cry43Cc1(Accession # KC156696); Cry43-like (Accession # BAD15305); Cry44Aa(Accession # BAD08532); Cry45Aa (Accession # BAD22577); Cry46Aa(Accession # BAC79010); Cry46Aa2 (Accession # BAG68906); Cry46Ab(Accession # BAD35170); Cry47Aa (Accession # AAY24695); Cry48Aa(Accession # CAJ18351); Cry48Aa2 (Accession # CAJ86545); Cry48Aa3(Accession # CAJ86546); Cry48Ab (Accession # CAJ86548); Cry48Ab2(Accession # CAJ86549); Cry49Aa (Accession # CAH56541); Cry49Aa2(Accession # CAJ86541); Cry49Aa3 (Accession # CAJ86543); Cry49Aa4(Accession # CAJ86544); Cry49Ab1 (Accession # CAJ86542); Cry50Aa1(Accession # BAE86999); Cry50Ba1 (Accession # GU446675); Cry50Ba2(Accession # GU446676); Cry51Aa1 (Accession # AB114444); Cry51Aa2(Accession # GU570697); Cry52Aa1 (Accession # EF613489); Cry52Ba1(Accession # FJ361760); Cry53Aa1 (Accession # EF633476); Cry53Ab1(Accession # FJ361759); Cry54Aa1 (Accession # ACA52194); Cry54Aa2(Accession # GQ140349); Cry54Ba1 (Accession # GU446677); Cry55Aa1(Accession # ABW88932); Cry54Ab1 (Accession # JQ916908); Cry55Aa2(Accession # AAE33526); Cry56Aa1 (Accession # ACU57499); Cry56Aa2(Accession # GQ483512); Cry56Aa3 (Accession # JX025567); Cry57Aa1(Accession # ANC87261); Cry58Aa1 (Accession # ANC87260); Cry59Ba1(Accession # JN790647); Cry59Aa1 (Accession # ACR43758); Cry60Aa1(Accession # ACU24782); Cry60Aa2 (Accession # EAO57254); Cry60Aa3(Accession # EEM99278); Cry60Ba1 (Accession # GU810818); Cry60Ba2(Accession # EAO57253); Cry60Ba3 (Accession # EEM99279); Cry61Aa1(Accession # HM035087); Cry61Aa2 (Accession # HM132125); Cry61Aa3(Accession # EEM19308); Cry62Aa1 (Accession # HM054509); Cry63Aa1(Accession # BA144028); Cry64Aa1 (Accession # BAJ05397); Cry65Aa1(Accession # HM461868); Cry65Aa2 (Accession # ZP_04123838); Cry66Aa1(Accession # HM485581); Cry66Aa2 (Accession # ZP_04099945); Cry67Aa1(Accession # HM485582); Cry67Aa2 (Accession # ZP_04148882); Cry68Aa1(Accession # HQ113114); Cry69Aa1 (Accession # HQ401006); Cry69Aa2(Accession # JQ821388); Cry69Ab1 (Accession # JN209957); Cry70Aa1(Accession # JN646781); Cry70Ba1 (Accession # ADO51070); Cry70Bb1(Accession # EEL67276); Cry71Aa1 (Accession # JX025568); Cry72Aa1(Accession # JX025569); Cyt1Aa (GenBank Accession Number X03182); Cyt1Ab(GenBank Accession Number X98793); Cyt1B (GenBank Accession NumberU37196); Cyt2A (GenBank Accession Number Z14147); and Cyt2B (GenBankAccession Number U52043).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275, 7,858,849 8,530,411, 8,575,433,and 8,686,233; a DIG-3 or DIG-11 toxin (N-terminal deletion of α-helix 1and/or α-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S.Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B of U.S. patentapplication Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1Fof U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat.Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Abprotein of U.S. Pat. No. 7,064,249); a Cry3A protein including but notlimited to an engineered hybrid insecticidal protein (eHIP) created byfusing unique combinations of variable regions and conserved blocks ofat least two different Cry proteins (US Patent Application PublicationNumber 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein;Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943,7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such assuch as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9Ffamilies, including but not limited to the Cry9D protein of U.S. Pat.No. 8,802,933 and the Cry9B protein of U.S. Pat. No. 8,802,934; a Cry15protein of Naimov, et al., (2008) Applied and EnvironmentalMicrobiology, 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat.Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 proteinof U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107and 7,504,229; a CryET33 and CryET34 homologs of US Patent PublicationNumber 2006/0191034, 2012/0278954, and PCT Publication Number WO2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; aTIC901 or related toxin; TIC807 of US Patent Application PublicationNumber 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128of PCT US 2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494,AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031,AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018,AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032;AXMI-003 of WO 2005/021585; AXMI-008 of US Patent ApplicationPublication Number 2004/0250311; AXMI-006 of US Patent ApplicationPublication Number 2004/0216186; AXMI-007 of US Patent ApplicationPublication Number 2004/0210965; AXMI-009 of US Patent ApplicationNumber 2004/0210964; AXMI-014 of US Patent Application PublicationNumber 2004/0197917; AXMI-004 of US Patent Application PublicationNumber 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US PatentApplication Publication Number 2011/0023184; AXMI-011, AXMI-012,AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033,AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of USPatent Application Publication Number 2011/0263488; AXMI-R1 and relatedproteins of US Patent Application Publication Number 2010/0197592;AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248;AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230and AXMI231 of WO 2011/103247 and U.S. Pat. No. 8,759,619; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US PatentApplication Publication Number 2010/0298211; AXMI-066 and AXMI-076 of USPatent Application Publication Number 2009/0144852; AXMI128, AXMI130,AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146,AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157,AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170,AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178,AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188,AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082,AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101,AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111,AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121,AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129,AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of USPatent Application Publication Number 2010/0005543, AXMI270 of US PatentApplication Publication US20140223598, AXMI279 of US Patent ApplicationPublication US20140223599, cry proteins such as Cry1A and Cry3A havingmodified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aaand Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 ofUS Patent Application Publication Number 2011/0064710. Other Cryproteins are well known to one skilled in the art (see, Crickmore, etal., “Bacillus thuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix). The insecticidal activity ofCry proteins is well known to one skilled in the art (for review, see,van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to plantsexpressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2,Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A,mCry3A, Cry9c and CBI-Bt have received regulatory approval (see,Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GMCrop Database Center for Environmental Risk Assessment (CERA), ILSIResearch Foundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database, which can be accessed onthe world-wide web using the “www” prefix). More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F(US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa(US2012/0317681); Cry1DA & Cry1BE (US2012/0331590); Cry1DA & Cry1Fa(US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa andCry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269);Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); Cry1Ab & Cry1F(US20140182018); and Cry3A and Cry1Ab or Vip3Aa (US20130116170).Pesticidal proteins also include insecticidal lipases including lipidacyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidasessuch as from Streptomyces (Purcell et al. (1993) Biochem Biophys ResCommun 15:1406-1413). Pesticidal proteins also include VIP (vegetativeinsecticidal proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,2796,137,033, 7,244,820, 7,615,686, and 8,237,020 and the like. Other VIPproteins are well known to one skilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but not limited to lycotoxin-1peptides and mutants thereof (U.S. Pat. No. 8,334,366).

In some embodiments the PIP-72 polypeptides include amino acid sequencesdeduced from the full-length nucleic acid sequences disclosed herein andamino acid sequences that are shorter than the full-length sequences,either due to the use of an alternate downstream start site or due toprocessing that produces a shorter protein having pesticidal activity.Processing may occur in the organism the protein is expressed in or inthe pest after ingestion of the protein.

Thus, provided herein are novel isolated or recombinant nucleic acidsequences that confer pesticidal activity. Also provided are the aminoacid sequences of PIP-72 polypeptides. The protein resulting fromtranslation of these PIP-72 polypeptide genes allows cells to control orkill pests that ingest it.

Bacterial Strains

One aspect pertains to bacterial strains that express a PIP-72polypeptide. In some embodiments the bacterial strain is a Halomonas,Photorhabdus, Xenorhabdus, Burkholderia, Paludibacterium or Pseudomonasspecies. In some embodiments the bacterial strain is a Halomonasanticariensis, Photorhabdus luminescens, Xenorhabdus bovienii,Burkholderia pseudomallei, Burkholderia multivorans, Burkholderiathailandensis, Paludibacterium yongneupense, Pseudomonas rhodesiae;Pseudomonas entomophila, Pseudomonas chlororaphis; Pseudomonas mandelii;Pseudomonas congelans; Pseudomonas mandelii; Pseudomonasplecoglossicida, Pseudomonas protegens, Pseudomonas ficuserectae;Pseudomonas mosselii or Pseudomonas brassicacearum strain. In someembodiments the bacterial strain is a biologically pure culture of aPseudomonas chlororaphis strain SS143D5, deposited on Feb. 7, 2013 underaccession # NRRL B-50810 with the Agricultural Research Service CultureCollection (NRRL), 1815 North University Street, Peoria, Ill. 61604,(nrrl.ncaur.usda.gov, which can be accessed on the world-wide web usingthe “www” prefix). The deposit will be maintained under the terms of theBudapest Treaty on the International Recognition of the Deposit ofMicroorganisms for the Purposes of Patent Procedure. These deposits weremade merely as a convenience for those of skill in the art and are notan admission that a deposit is required under 35 U.S.C. § 112. Access tothis deposit will be available during the pendency of the application tothe Commissioner of Patents and Trademarks and persons determined by theCommissioner to be entitled thereto upon request. Upon allowance of anyclaims in the application, the Applicant(s) will make available to thepublic, pursuant to 37 C.F.R. § 1.808, sample(s) of the deposit of withthe Agricultural Research Service Culture Collection (NRRL), 1815 NorthUniversity Street, Peoria, Ill. 61604. This deposit will be maintainedin the NRRL depository, which is a public depository, for a period of 30years or 5 years after the most recent request or for the enforceablelife of the patent, whichever is longer, and will be replaced if itbecomes nonviable during that period. The deposits will irrevocably andwithout restriction or condition be available to the public uponissuance of a patent. Additionally, Applicant(s) have satisfied all therequirements of 37 C.F.R. §§ 1.801-1.809, including providing anindication of the viability of the sample upon deposit. Applicant(s)have no authority to waive any restrictions imposed by law on thetransfer of biological material or its transportation in commerce.Applicant(s) do not waive any infringement of their rights granted underthis patent. However, it should be understood that the availability of adeposit does not constitute a license to practice the subject inventionin derogation of patent rights granted by government action.

Nucleic Acid Molecules, and Variants and Fragments Thereof

One aspect pertains to isolated or recombinant nucleic acid moleculescomprising nucleic acid sequences encoding PIP-72 polypeptides orbiologically active portions thereof, as well as nucleic acid moleculessufficient for use as hybridization probes to identify nucleic acidmolecules encoding proteins with regions of sequence homology. As usedherein, the term “nucleic acid molecule” refers to DNA molecules (e.g.,recombinant DNA, cDNA, genomic DNA, plastid DNA, mitochondrial DNA) andRNA molecules (e.g., mRNA) and analogs of the DNA or RNA generated usingnucleotide analogs. The nucleic acid molecule can be single-stranded ordouble-stranded, but preferably is double-stranded DNA.

An “isolated” nucleic acid molecule (or DNA) is used herein to refer toa nucleic acid sequence (or DNA) that is no longer in its naturalenvironment, for example in vitro. A “recombinant” nucleic acid molecule(or DNA) is used herein to refer to a nucleic acid sequence (or DNA)that is in a recombinant bacterial or plant host cell. In someembodiments, an “isolated” or “recombinant” nucleic acid is free ofsequences (preferably protein encoding sequences) that naturally flankthe nucleic acid (i.e., sequences located at the 5′ and 3′ ends of thenucleic acid) in the genomic DNA of the organism from which the nucleicacid is derived. For purposes of the disclosure, “isolated” or“recombinant” when used to refer to nucleic acid molecules excludesisolated chromosomes. For example, in various embodiments, therecombinant nucleic acid molecule encoding a PIP-72 polypeptide cancontain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kbof nucleic acid sequences that naturally flank the nucleic acid moleculein genomic DNA of the cell from which the nucleic acid is derived.

In some embodiments an isolated nucleic acid molecule encoding a PIP-72polypeptide has one or more change in the nucleic acid sequence comparedto the native or genomic nucleic acid sequence. In some embodiments thechange in the native or genomic nucleic acid sequence includes but isnot limited to: changes in the nucleic acid sequence due to thedegeneracy of the genetic code; changes in the nucleic acid sequence dueto the amino acid substitution, insertion, deletion and/or additioncompared to the native or genomic sequence; removal of one or moreintron; deletion of one or more upstream or downstream regulatoryregions; and deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence. In some embodiments the nucleicacid molecule encoding a PIP-72 polypeptide is a non-genomic sequence.

A variety of polynucleotides that encode a PIP-72 polypeptides orrelated proteins are contemplated. Such polynucleotides are useful forproduction of PIP-72 polypeptides in host cells when operably linked tosuitable promoter, transcription termination and/or polyadenylationsequences. Such polynucleotides are also useful as probes for isolatinghomologous or substantially homologous polynucleotides that encodePIP-72 polypeptides or related proteins.

Sources of polynucleotides that encode PIP-72 polypeptides or relatedproteins include but not limited to Halomonas anticariensis,Photorhabdus luminescens, Xenorhabdus bovienii, Burkholderiapseudomallei, Burkholderia multivorans, Burkholderia thailandensis,Paludibacterium yongneupense, Pseudomonas rhodesiae; Pseudomonasentomophila, Pseudomonas chlororaphis; Pseudomonas mandelii; Pseudomonascongelans; Pseudomonas mandelii; Pseudomonas plecoglossicida,Pseudomonas protegens, Pseudomonas ficuserectae; Pseudomonas mosselii orPseudomonas brassicacearum strain. Sources of polynucleotides thatencode PIP-72 polypeptides or related proteins include but not limitedto: a Pseudomonas chlororaphis strain which contains the PIP-72Aapolynucleotide of SEQ ID NO: 1 encoding the PIP-72Aa polypeptide of SEQID NO: 2; a Pseudomonas rhodesiae strain which contains the PIP-72Bapolynucleotide of SEQ ID NO: 3 encoding the PIP-72Ba polypeptide of SEQID NO: 4; a Pseudomonas chlororaphis strain which contains the PIP-72Capolynucleotide of SEQ ID NO: 5 encoding the PIP-72Ca polypeptide of SEQID NO: 6; a Pseudomonas mandelii strain which contains the PIP-72Cbpolynucleotide of SEQ ID NO: 7 encoding the PIP-72Cb polypeptide of SEQID NO: 8; a Pseudomonas congelans strain which contains the PIP-72 Dapolynucleotide of SEQ ID NO: 9 encoding the PIP-72 Da polypeptide of SEQID NO: 10; a Pseudomonas mandelii strain which contains the PIP-72Dbpolynucleotide of SEQ ID NO: 11 encoding the PIP-72Db polypeptide of SEQID NO: 12; a Pseudomonas ficuserectae strain which contains the PIP-72Dcpolynucleotide of SEQ ID NO: 13 encoding the PIP-72Dc polypeptide of SEQID NO: 14; a Pseudomonas mosselii strain which contains the PIP-72Fapolynucleotide of SEQ ID NO: 17 encoding the PIP-72Fa polypeptide of SEQID NO: 18; a Pseudomonas chlororaphis strain which contains the PIP-72Ffpolynucleotide of SEQ ID NO: 27 encoding the PIP-72Ff polypeptide of SEQID NO: 28; a Pseudomonas chlororaphis strain which contains the PIP-72Gbpolynucleotide of SEQ ID NO: 31 encoding the PIP-72Gb polypeptide of SEQID NO: 32; a Pseudomonas chlororaphis strain which contains the PIP-72Abpolynucleotide of SEQ ID NO: 949 encoding the PIP-72Ab polypeptide ofSEQ ID NO: 927; a Pseudomonas brassicacearum strain which contains thePIP-72Bb polynucleotide of SEQ ID NO: 950 encoding the PIP-72Abpolypeptide of SEQ ID NO: 928; a Pseudomonas entomophila strain whichcontains the PIP-72Fh polynucleotide of SEQ ID NO: 954 encoding thePIP-72AFh polypeptide of SEQ ID NO: 932; a Pseudomonas entomophilastrain which contains the PIP-72Fh polynucleotide of SEQ ID NO: 955encoding the PIP-72AFh polypeptide of SEQ ID NO: 933; a Pseudomonaschlororaphis strain which contains the PIP-72Fj polynucleotide of SEQ IDNO: 956 encoding the PIP-72Fj polypeptide of SEQ ID NO: 934; aPseudomonas chlororaphis strain which contains the PIP-72Fkpolynucleotide of SEQ ID NO: 957 encoding the PIP-72Fk polypeptide ofSEQ ID NO: 935; a Burkholderia multivorans strain which contains thePIP-72FI polynucleotide of SEQ ID NO: 958 encoding the PIP-72FIpolypeptide of SEQ ID NO: 936; a Pseudomonas chlororaphis strain whichcontains the PIP-72Gg polynucleotide of SEQ ID NO: 961 encoding thePIP-72Gg polypeptide of SEQ ID NO: 939; a Pseudomonas chlororaphisstrain which contains the PIP-72Gh polynucleotide of SEQ ID NO: 962encoding the PIP-72Gh polypeptide of SEQ ID NO: 940; a Pseudomonasmosselii strain which contains the PIP-72Gi polynucleotide of SEQ ID NO:963 encoding the PIP-72Gi polypeptide of SEQ ID NO: 941; a Pseudomonasprotegens strain which contains the PIP-72Gk polynucleotide of SEQ IDNO: 965 encoding the PIP-72Gk polypeptide of SEQ ID NO: 943; aPseudomonas plecoglossicida strain which contains the PIP-72GIpolynucleotide of SEQ ID NO: 966 encoding the PIP-72GI polypeptide ofSEQ ID NO: 944; and a Pseudomonas chlororaphis strain which contains thePIP-72Gn polynucleotide of SEQ ID NO: 968 encoding the PIP-72Gnpolypeptide of SEQ ID NO: 946. These polynucleotide sequences wereisolated from a Halomonas, Photorhabdus, Xenorhabdus, Burkholderia,Paludibacterium or Pseudomonas host and are thus suitable for expressionof the encoded PIP-72 polypeptide in other bacterial hosts. For example,SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9,SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27 and SEQ IDNO: 31, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 955, SEQ ID NO: 956,SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ IDNO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 968can be used to express PIP-72 polypeptides in bacterial hosts thatinclude but are not limited to Agrobacterium, Bacillus, Escherichia,Salmonella, Pseudomonas and Rhizobium bacterial host cells. Thepolynucleotides are also useful as probes for isolating homologous orsubstantially homologous polynucleotides that encode PIP-72 polypeptidesor related proteins. Such probes can be used to identify homologous orsubstantially homologous polynucleotides derived from Halomonas,Photorhabdus, Xenorhabdus, Burkholderia, Paludibacterium, Pseudomonas orother related bacteria.

Polynucleotides that encode a PIP-72 polypeptide can also be synthesizedde novo from a PIP-72 polypeptide sequence. The sequence of thepolynucleotide gene can be deduced from a PIP-72 polypeptide sequencethrough use of the genetic code. Computer programs such as“BackTranslate” (GCG™ Package, Acclerys, Inc. San Diego, Calif.) can beused to convert a peptide sequence to the corresponding nucleotidesequence encoding the peptide. Examples of PIP-72 polypeptide sequencesthat can be used to obtain corresponding nucleotide encoding sequencesinclude, but are not limited to, the PIP-72 polypeptide of sequence SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28 and SEQ ID NO:32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO:940, SEQ ID NO: 941SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQID NO: 946. Furthermore, synthetic PIP-72 polynucleotide sequences ofthe disclosure can be designed so that they will be expressed in plants.U.S. Pat. No. 5,500,365 describes a method for synthesizing plant genesto improve the expression level of the protein encoded by thesynthesized gene. This method relates to the modification of thestructural gene sequences of the exogenous transgene, to cause them tobe more efficiently transcribed, processed, translated and expressed bythe plant. Features of genes that are expressed well in plants includeelimination of sequences that can cause undesired intron splicing orpolyadenylation in the coding region of a gene transcript whileretaining substantially the amino acid sequence of the toxic portion ofthe insecticidal protein. A similar method for obtaining enhancedexpression of transgenes in monocotyledonous plants is disclosed in U.S.Pat. No. 5,689,052.

In some embodiments the nucleic acid molecule encoding a PIP-72polypeptide is a polynucleotide having the sequence set forth in SEQ IDNO: 1; SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQID NO: 949, SEQ ID NO: 950, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO:957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 968, andvariants, fragments and complements thereof. “Complement” is used hereinto refer to a nucleic acid sequence that is sufficiently complementaryto a given nucleic acid sequence such that it can hybridize to the givennucleic acid sequence to thereby form a stable duplex. “Polynucleotidesequence variants” is used herein to refer to a nucleic acid sequencethat except for the degeneracy of the genetic code encodes the samepolypeptide.

In some embodiments a nucleic acid molecule encoding the PIP-72polypeptide is a non-genomic nucleic acid sequence. As used herein a“non-genomic nucleic acid sequence” or “non-genomic nucleic acidmolecule” refers to a nucleic acid molecule that has one or more changein the nucleic acid sequence compared to a native or genomic nucleicacid sequence. In some embodiments the change to a native or genomicnucleic acid molecule includes but is not limited to: changes in thenucleic acid sequence due to the degeneracy of the genetic code; codonoptimization of the nucleic acid sequence for expression in plants;changes in the nucleic acid sequence to introduce at least one aminoacid substitution, insertion, deletion and/or addition compared to thenative or genomic sequence; removal of one or more intron associatedwith the genomic nucleic acid sequence; insertion of one or moreheterologous introns; deletion of one or more upstream or downstreamregulatory regions associated with the genomic nucleic acid sequence;insertion of one or more heterologous upstream or downstream regulatoryregions; deletion of the 5′ and/or 3′ untranslated region associatedwith the genomic nucleic acid sequence; insertion of a heterologous 5′and/or 3′ untranslated region; and modification of a polyadenylationsite. In some embodiments the non-genomic nucleic acid molecule is acDNA. In some embodiments the non-genomic nucleic acid molecule is asynthetic nucleic acid sequence. In some embodiments the non-genomicnucleic molecule is not the nucleic acid sequence of SEQ ID NO: 1, SEQID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQ ID NO: 949,SEQ ID NO: 950, SEQ ID NO: 955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ IDNO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965,SEQ ID NO: 966, SEQ ID NO: 967, SEQ ID NO: 968.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28,SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ IDNO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939,SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ IDNO: 945 or SEQ ID NO: 946, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 2, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule comprising anamino acid sequence having at least 50%, 51%, 52%, 53%, 54%, 55%, 56%,57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%,71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or99% identity to the amino acid sequence of SEQ ID NO: 4, wherein thepolypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 6, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 8, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%,63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%,77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%,91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 10, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 12, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 14, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 18, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 28, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 32, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 927, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 928, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 932, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 933, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 934, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 935, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 936, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 939, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 940, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 941, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 943, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 944, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 945, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%,78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the amino acidsequence of SEQ ID NO: 946, wherein the polypeptide has pesticidalactivity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928,SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ IDNO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943,SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO:927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO:941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946,and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 2, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 2and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 4, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 4and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 6, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 6and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 8, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 8and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 10, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 10and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 12, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 12and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence of SEQ ID NO: 14, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 14and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 60%identity to the amino acid sequence of SEQ ID NO: 18, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 18and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 85%identity to the amino acid sequence of SEQ ID NO: 28, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO: 28and the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 32, wherein the PIP-72polypeptide has at least one amino acid change compared to SEQ ID NO:32, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 927, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 927, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 928, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 928, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 932, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 932, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 933, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 933, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 934, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 934, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 935, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 935, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 936, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 936, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 939, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 939, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 940, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 940, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 941, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 941, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 943, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 943, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 944, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 944, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 945, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 945, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence having at least 95%identity to the amino acid sequence of SEQ ID NO: 946, wherein thePIP-72 polypeptide has at least one amino acid change compared to SEQ IDNO: 946, and wherein the PIP-72 polypeptide has pesticidal activity.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO:927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO:941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO:927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO:941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 2having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 4having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 4.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 6having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 6.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 8having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 8.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 10having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 10.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 12having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 12.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 14having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO: 14.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 18having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35 or 36amino acid substitutions compared to the native amino acid at thecorresponding position of SEQ ID NO: 18.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 28having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or 14 amino acidsubstitutions compared to the native amino acid at the correspondingposition of SEQ ID NO: 28.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 32having 1, 2, 3, 4 or 5 amino acid substitutions compared to the nativeamino acid at the corresponding position of SEQ ID NO: 32.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 927having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:927.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 928having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:928.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 932having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:932.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 933having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:933.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 934having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:934.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 935having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:935.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 936having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:936.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 939having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:939.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 940having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:940.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 941having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:941.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 943having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:943.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 944having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:944.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 945having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:945.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 946having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions comparedto the native amino acid at the corresponding position of SEQ ID NO:946.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 846having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions, atpositions designated by Xaa, compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 847having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions, atpositions designated by Xaa, compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 848having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions, atpositions designated by Xaa, compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments the non-genomic nucleic acid molecule encodes aPIP-72 polypeptide comprising an amino acid sequence of SEQ ID NO: 849having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36,37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acid substitutions, atpositions designated by Xaa, compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments the nucleic acid molecule encodes a PIP-72polypeptide comprising an amino acid sequence of SEQ ID NO: 846, whereinXaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu, Asn, Arg,Ser, Thr, Val, Trp or Tyr; Xaa at position 3 is Ile or Trp; Xaa atposition 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser, Val, Trpor Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile or Tyr; Xaa atposition 6 is Thr, Ala, Cys, Phe, Gly, His, Ile, Lys, Met, Pro, Gln,Arg, Ser, Trp or Tyr; Xaa at position 7 is Asn, Ala or Val; Xaa atposition 8 is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met,Gln, Arg, Ser, Thr or Val; Xaa at position 9 is Ser, Ala, Cys, Gly orThr; Xaa at position 10 is Ser, Ala, Glu, Phe, Gly, His, Ile, Lys, Leu,Asn, Pro, Gln, Arg, Thr or Trp; Xaa at position 11 is Asn, Ala, Cys,Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val or Tyr; Xaaat position 12 is Pro, Ala, Cys, Asp, Glu, Gly, His, Lys, Leu, Asn, Gln,Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 13 is Ile, Asn, Gln orVal; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys or Gln; Xaa atposition 15 is Val, Ala, Cys, Ile, Met or Arg; Xaa at position 17 isIle, Glu or Val; Xaa at position 18 is Asn or Ser; Xaa at position 19 isHis, Ala, Glu, Lys, Leu, Pro, Arg, Ser or Tyr; Xaa at position 20 isTrp, Ala or Thr; Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr or Val; Xaa atposition 24 is Gly, Asp or Phe; Xaa at position 25 is Asp, Ala, Glu,Phe, Asn or Gln; Xaa at position 26 is Thr, Glu or Pro; Xaa at position27 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, Gln, Arg or Thr; Xaaat position 28 is Phe, Pro, Trp or Tyr; Xaa at position 29 is Phe, Ala,Cys, Ile, Leu, Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Ala,Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp or Tyr; Xaa at position 31 is Val, Ile or Leu; Xaa at position32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp or Tyr; Xaa at position 33 is Asn, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val or Tyr;Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr or Tyr; Xaa at position 35 is Lys, Ala, Cys, Asp, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa at position 36 isGln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr orVal; Xaa at position 37 is Glu, Ala, Cys, Asp, Phe, Gly, Ile, Lys, Leu,Met, Asn, Ser, Thr or Val; Xaa at position 38 is Thr, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr;Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; Xaa at position 42 is Ser, Ala, Cys, Asp, Glu, Phe, Gly,Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position44 is Ser, Ala, Asp, Glu, Gly, Leu, Met, Asn, Pro, Gln, Thr, Val or Tyr;Xaa at position 45 is Arg, Lys or Ser; Xaa at position 46 is Gly, Ala orGln; Xaa at position 47 is Phe, Cys, Val or Tyr; Xaa at position 48 isVal, Ile or Leu; Xaa at position 49 is Leu, Cys, Phe, Met, Arg or Tyr;Xaa at position 50 is Ser, Ala, Cys, Asp, Ile, Met, Pro, Gln, Thr orVal; Xaa at position 51 is Leu, Ala, Cys, Met or Val; Xaa at position 52is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Trp or Tyr;Xaa at position 53 is Lys, Ala, Cys, Asp, Glu, Phe, His, Ile, Leu, Met,Asn, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 54 is Asn, Cys,Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position 56is Ala, Gly, Leu, Asn, Pro, Gln, Arg, Ser or Thr; Xaa at position 57 isGln, Glu, Leu, Met, Ser or Thr; Xaa at position 58 is His, Ala, Asp,Phe, Leu, Met, Asn, Arg, Trp or Tyr; Xaa at position 60 is Tyr, Glu orPhe; Xaa at position 63 is Gln, Cys, Gly, Ile, Leu, Met, Asn, Thr, Valor Tyr; Xaa at position 64 is Ala, Phe, Gly, His, Arg, Ser or Tyr; Xaaat position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn,Thr or Val; Xaa at position 66 is Ser, Ala or Gly; Xaa at position 67 isLys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu or Val; Xaa atposition 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Cys or Ile; Xaa atposition 71 is Asp, Ala, Cys, Gly, His, Ile, Leu, Met, Asn, Ser, Thr,Val or Tyr; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys,Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 73 is Asn,Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Ser, Thr, Val or Tyr; Xaa atposition 74 is Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 75 is Val, Cys, Ile or Leu; Xaa atposition 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 77 is Asp Tyr; Xaa at position 78 isGln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr,Val or Tyr; Xaa at position 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe,His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp or Tyr; Xaa at position 80is Arg, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Val orTyr; Xaa at position 81 is Leu, Ala, Cys, Asp, Phe, Gly, His, Ile, Asn,Pro, Arg, Ser, Thr or Val; Xaa at position 82 is Ile, Ala, Leu, Met, Argor Val; Xaa at position 83 is Glu, Ala, Cys, Asp, Phe, Gly, His, Ile,Lys, Leu, Asn, Pro, Arg, Ser, Thr, Val or Tyr; Xaa at position 84 isPro, Ala, Cys, Glu, Ile, Ser, Val, Trp or Tyr; Xaa at position 85 isLeu, Cys, Gly or Val; and Xaa at position 86 is Ser, Ala, Ile, Thr orVal, and wherein 1 to 14 amino acids are optionally deleted from theN-terminus and/or C-terminus of the PIP-72 polypeptide.

In some embodiments the nucleic acid molecule encodes a PIP-72polypeptide comprising an amino acid sequence of SEQ ID NO: 847, whereinXaa at position 2 is Gly, Lys or Ala; Xaa at position 3 is Ile or Leu;Xaa at position 4 is Thr or Ser; Xaa at position 5 is Val or Ile; Xaa atposition 6 is Thr or Lys; Xaa at position 8 is Asn, Lys, Gly or Ser; Xaaat position 9 is Ser or Ala; Xaa at position 11 is Asn, Lys, His or Thr;Xaa at position 12 is Pro, Thr, Lys or Ser; Xaa at position 13 is Ile orVal; Xaa at position 14 is Glu or Asp; Xaa at position 15 is Val, Ala orIle; Xaa at position 16 is Ala or Ser; Xaa at position 17 is Ile or Val;Xaa at position 18 is Asn or Ser; Xaa at position 19 is His, Lys, Arg,Gln or Ala; Xaa at position 21 is Gly or Arg; Xaa at position 22 is Ser,Lys, Asn, Asp or Thr; Xaa at position 25 is Asp or Asn; Xaa at position26 is Thr or Asp; Xaa at position 27 is Ser, Thr, Asn or Lys; Xaa atposition 28 is Phe, Tyr or Pro; Xaa at position 29 is Phe or Tyr; Xaa atposition 30 is Ser, Gly or Lys; Xaa at position 31 is Val, Ile or Met;Xaa at position 32 is Gly, Ala or Asp; Xaa at position 33 is Asn, Ser,Gln or Pro; Xaa at position 35 is Lys, Glu or Ser; Xaa at position 36 isGln, Asn or Ser; Xaa at position 37 is Glu or Asp; Xaa at position 38 isThr or Ser; Xaa at position 42 is Ser or Asn; Xaa at position 44 is Ser,Asp, Ala or Leu; Xaa at position 47 is Phe or Tyr; Xaa at position 48 isLeu or Met; Xaa at position 49 is Leu or Met; Xaa at position 50 is Ser,Ala or Tyr; Xaa at position 51 is Leu or Val; Xaa at position 52 is Lysor Gln; Xaa at position 53 is Lys, Arg, Met or Leu; Xaa at position 54is Asn, Lys or Gly; Xaa at position 55 is Gly or Ser; Xaa at position 56is Ala, Thr, Gln or Ser; Xaa at position 57 is Gln, Val or Ala; Xaa atposition 58 is His, Ala, Lys, Tyr or Thr; Xaa at position 59 is Pro orThr; Xaa at position 62 is Val or Ile; Xaa at position 63 is Gln, Ser orLeu; Xaa at position 64 is Ala, Gln or Ser; Xaa at position 65 is Ser orThr; Xaa at position 67 is Lys, Gln, Arg or Asn; Xaa at position 69 isGlu, Lys or Val; Xaa at position 70 is Val or Ile; Xaa at position 71 isAsp, Glu or Tyr; Xaa at position 72 is Asn, His, Ser or Asp; Xaa atposition 73 is Asn, Ser or Asp; Xaa at position 74 is Ala, Thr, Met, Ileor Lys; Xaa at position 76 is Lys or Thr; Xaa at position 78 is Gln, Hisor Ser; Xaa at position 80 is Arg, Glu or Gln; Xaa at position 81 isLeu, Pro, Ala or Thr; Xaa at position 82 is Ile or Leu; Xaa at position83 is Glu, His, Asn, Gln or Leu; Xaa at position 85 is Leu, Val or Ala;and Xaa at position 86 is Ser, Ala, Tyr or Asn, and wherein, 1 to 14amino acids are optionally deleted from the N-terminus and/or C-terminusof the PIP-72 polypeptide and/or an amino acid is inserted betweenresidue 24 and 25 relative to SEQ ID NO: 847.

In some embodiments the nucleic acid molecule encodes a PIP-72polypeptide comprising an amino acid sequence of SEQ ID NO: 848, whereinXaa at position 2 is Gly, Lys, Ala or Arg; Xaa at position 3 is Ile, Leuor Val; Xaa at position 4 is Thr or Ser; Xaa at position 5 is Val, Ileor Leu; Xaa at position 6 is Thr, Lys, Ser or Arg; Xaa at position 8 isAsn, Lys, Gly, Ser, Gln, Arg, Thr or Ala; Xaa at position 9 is Ser, Alaor Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, His or Ser; Xaaat position 12 is Pro, Thr, Lys, Ser or Arg; Xaa at position 13 is Ile,Val or Leu; Xaa at position 14 is Glu or Asp; Xaa at position 15 is Val,Ala, Ile or Leu; Xaa at position 16 is Ala or Ser; Xaa at position 17 isIle, Val or Leu; Xaa at position 18 is Asn, Ser, Gln or Thr; Xaa atposition 19 is His, Lys, Ala, Gln, Asn or Arg; Xaa at position 21 isGly, Arg or Lys; Xaa at position 22 is Ser, Lys, Asn, Thr, Arg, Asp, Gluor Gln; Xaa at position 25 is Asp, Asn, Glu or Gln; Xaa at position 26is Thr, Asp, Ser or Glu; Xaa at position 27 is Ser, Thr, Lys, Asn, Glnor Arg; Xaa at position 28 is Phe, Tyr, Pro or Trp; Xaa at position 29is Phe, Tyr or Trp; Xaa at position 30 is Ser, Gly, Lys, Thr or Arg; Xaaat position 31 is Val, Ile, Met or Leu; Xaa at position 32 is Gly, Ala,Asp or Glu; Xaa at position 33 is Asn, Ser, Gln, Pro or Thr; Xaa atposition 35 is Lys, Glu, Ser, Arg or Thr; Xaa at position 36 is Gln,Ser, Asn or Thr; Xaa at position 37 is Glu or Asp; Xaa at position 38 isThr or Ser; Xaa at position 42 is Ser, Asn, Thr or Gln; Xaa at position44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile or Val; Xaa at position 47 isPhe, Tyr or Trp; Xaa at position 48 is Leu, Met, Ile or Val; Xaa atposition 49 is Leu, Met, Ile or Val; Xaa at position 50 is Ser, Ala, Tyror Thr; Xaa at position 51 is Leu, Val or Ile; Xaa at position 52 isLys, Gln, Arg or Asn; Xaa at position 53 is Lys, Arg, Met, Leu, Ile orVal; Xaa at position 54 is Asn, Lys, Gly, Gln or Arg; Xaa at position 55is Gly, Ser or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser or Asn; Xaaat position 57 is Gln, Val, Ala, Asn, Leu or Ile; Xaa at position 58 isHis, Ala, Lys, Tyr or Thr; Xaa at position 59 is Pro, Thr or Ser; Xaa atposition 62 is Val, Ile or Leu; Xaa at position 63 is Gln, Ser, Leu,Asn, Thr, Ile or Val; Xaa at position 64 is Ala, Gln, Ser, Asn or Thr;Xaa at position 65 is Ser or Thr; Xaa at position 67 is Lys, Gln, Asn orArg; Xaa at position 69 is Glu, Val, Asp, Lys, Arg, Ile or Leu; Xaa atposition 70 is Val, Ile or Leu; Xaa at position 71 is Asp, Glu, Tyr orTrp; Xaa at position 72 is Asn, His, Ser, Asp, Gln, Thr or Glu; Xaa atposition 73 is Asn, Ser, Asp, Gln, Thr or Glu; Xaa at position 74 isAla, Thr, Met, Ile, Lys, Ser, Leu, Val or Arg; Xaa at position 76 isLys, Thr, Arg or Ser; Xaa at position 78 is Gln, His, Ser, Asn or Thr;Xaa at position 80 is Arg, Glu, Gln, Lys, Asp or Asn; Xaa at position 81is Leu, Pro, Thr, Ile, Val, Ala or Ser; Xaa at position 82 is Ile, Leuor Val; Xaa at position 83 is Glu, His, Asn, Leu, Gln, Ile or Val; Xaaat position 85 is Leu, Val or Ala; and Xaa at position 86 is Ser, Ala,Tyr, Asn or Thr, and wherein, 1 to 14 amino acids are optionally deletedfrom the N-terminus and/or C-terminus of the PIP-72 polypeptide and/oran amino acid is inserted between residue 24 and 25 relative to SEQ IDNO: 848.

In some embodiments the nucleic acid molecule encodes a PIP-72polypeptide comprising an amino acid sequence of SEQ ID NO: 849, whereinXaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu, Asn, Arg,Ser, Thr, Val, Trp or Tyr; Xaa at position 3 is Ile, Leu, Val or Trp;Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser,Val, Trp or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile, Leuor Tyr; Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, Ile, Lys,Met, Pro, Gln, Arg, Ser, Trp or Tyr; Xaa at position 7 is Asn, Ala orVal; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thr, Ala, Cys,Asp, Glu, His, Ile, Leu, Met or Val; Xaa at position 9 is Ser, Ala, Cys,Gly or Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, Ser, Ala,Cys, Asp, Glu, Gly, His, Ile, Leu, Met, Val or Tyr; Xaa at position 12is Pro, Thr, Lys, Ser, Arg, Ala, Cys, Asp, Glu, Gly, His, Leu, Asn, Gln,Arg, Val, Trp or Tyr; Xaa at position 13 is Ile, Asn, Gln, Leu or Val;Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, Asp or Gln; Xaa atposition 15 is Val, Ala, Ile, Leu, Cys, Met or Arg; Xaa at position 16is Ala or Ser; Xaa at position 17 is Ile, Glu, Leu or Val; Xaa atposition 18 is Asn, Gln, Thr or Ser; Xaa at position 19 is His, Lys,Ala, Arg, Glu, Leu, Pro, Ser or Tyr; Xaa at position 20 is Trp, Ala orThr; Xaa at position 21 is Gly, Arg or Lys; Xaa at position 22 is Ser,Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val or Tyr; Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn,Gln, Ser, Thr or Val; Xaa at position 24 is Gly, Asp or Phe; Xaa atposition 25 is Asp, Ala, Glu, Phe, Asn or Gln; Xaa at position 26 isThr, Glu, Asp, Ser or Pro; Xaa at position 27 is Ser, Thr, Lys, Arg,Ala, Cys, Asp, Glu, Phe, Gly, His, Asn or Gln; Xaa at position 28 isPhe, Tyr, Pro or Trp; Xaa at position 29 is Phe, Ala, Cys, Ile, Leu,Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Gly, Lys, Thr, Arg,Ala, Cys, Asp, Glu, Phe, His, Leu, Met, Asn, Pro, Gln, Val, Trp or Tyr;Xaa at position 31 is Val, Ile, Met or Leu; Xaa at position 32 is Gly,Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 33 is Asn, Ser, Gln, Pro, Thr, Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Arg, Val or Tyr; Xaa atposition 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr or Tyr; Xaa at position 35 is Lys, Glu, Ala, Cys, Asp, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa at position 36 isGln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr orVal; Xaa at position 37 is Glu, Asp, Ala, Cys, Phe, Gly, Ile, Lys, Leu,Met, Asn, Ser, Thr or Val; Xaa at position 38 is Thr, Ser, Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Val, Trp or Tyr;Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; Xaa at position 42 is Ser, Asn, Thr, Ala, Cys, Asp, Glu,Phe, Gly, Ile, Lys, Leu, Met, Arg, Val, Trp, Tyr or Gln; Xaa at position44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, Ala, Gly, Leu, Met, Asn, Pro,Gln, Val, Tyr or Val; Xaa at position 45 is Arg, Lys or Ser; Xaa atposition 46 is Gly, Ala or Gln; Xaa at position 47 is Phe, Tyr Cys, Valor Trp; Xaa at position 48 is Leu, Met, Ile, Cys, Phe, Met, Arg, Tyr orVal; Xaa at position 49 is Leu, Met, Ile or Val; Xaa at position 50 isSer, Ala, Tyr, Cys, Asp, Ile, Met, Pro, Gln, Val or Thr; Xaa at position51 is Leu, Val, Ala, Cys, Met or Ile; Xaa at position 52 is Lys, Cys,Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Gln, Trp or Tyr; Xaa atposition 53 is Lys, Arg, Met, Leu, Ile, Ala, Cys, Asp, Glu, Phe, His,Asn, Gln, Ser, Thr, Tyr or Val; Xaa at position 54 is Asn, Cys, Asp,Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position 55 isGly, Ser or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, Gly, Leu,Pro, Arg or Asn; Xaa at position 57 is Gln, Glu, Leu, Met, Ser, Val,Ala, Asn, Ile or Thr; Xaa at position 58 is His, Ala, Lys, Asp, Phe,Leu, Met, Asn, Arg, Trp, Tyr or Thr; Xaa at position 59 is Pro, Thr orSer; Xaa at position 60 is Tyr, Glu or Phe; Xaa at position 62 is Val,Ile or Leu; Xaa at position 63 is Gln, Ser, Cys, Gly, Ile, Leu, Met,Asn, Thr, Val or Tyr; Xaa at position 64 is Ala, Gln, Asn, Phe, Gly,His, Arg, Ser or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Leu, Asn, Val or Thr; Xaa at position 66 is Ser, Alaor Gly; Xaa at position 67 is Lys, Gln, Asn or Arg; Xaa at position 67is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu or Val; Xaa atposition 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Ile, Cys or Leu; Xaa atposition 71 is Asp, Glu, Tyr, Ala, Cys, Gly, His, Ile, Leu, Met, Asn,Ser, Thr, Val or Trp; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu,Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, His or Trp; Xaa at position73 is Asn, Ser, Asp, Gln, Thr, Ala, Cys, Phe, Gly, His, Ile, Leu, Val,Tyr or Glu; Xaa at position 74 is Ala, Thr, Met, Ile, Lys, Ser, Leu,Val, Cys, Asp, Phe, Gly, His, Asn, Gln, Tyr or Arg; Xaa at position 75is Val, Cys, Ile or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His,Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 77 is AspTyr; Xaa at position 78 is Gln, His, Ser, Asn, Ala, Cys, Asp, Phe, Gly,Ile, Leu, Met, Asn, Arg, Val, Tyr or Thr; Xaa at position 79 is Gly,Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Trp or Tyr; Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, Ala, Cys,Phe, Gly, His, Ile, Leu, Ser, Thr, Val, Tyr or Asn; Xaa at position 81is Leu, Pro, Thr, Ile, Val, Ala, Cys, Asp, Phe, Gly, His or Ser; Xaa atposition 82 is Ile, Ala, Leu, Met, Arg and Val; Xaa at position 83 isGlu, His, Asn, Leu, Gln, Ile, Ala, Cys, Asp, Phe, Gly, Lys, Pro, Arg,Ser, Thr, Tyr or Val; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile,Ser, Val, Trp or Tyr; Xaa at position 85 is Leu, Val, Cys, Gly or Ala;and Xaa at position 86 is Ser, Ala, Tyr, Asn, Ile, Val or Thr, andwherein, 1 to 14 amino acids are optionally deleted from the N-terminusand/or C-terminus of the PIP-72 polypeptide and/or an amino acid isinserted between residue 24 and 25 relative to SEQ ID NO: 849.

In some embodiments the nucleic acid molecules encode a PIP-72polypeptide comprising an amino acid motif as represented by positions37-51 of SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848 or SEQ ID NO:849.

In some embodiments the nucleic acid molecules encode a PIP-72polypeptide comprising an amino acid sequence having at least 50%identity to the amino acid sequence set forth in SEQ ID NO: 2

In some embodiments exemplary nucleic acid molecules encode a PIP-72polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8,SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO:28, SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one ofSEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ IDNO: 852, any one of SEQ ID NO: 903-914, SEQ ID NO: 927, SEQ ID NO: 928,SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ IDNO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943,SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, as well as amino acidsubstitutions deletions, insertions and fragments thereof andcombinations thereof.

In some embodiments the nucleic acid molecules encode a PIP-72polypeptide of Table 14, Table 17, Table 20, Table 23, Table 24, Table26, Table 28, and/or Table 29, combinations of the amino acidsubstitutions thereof, and deletions and/or insertions thereof.

Also provided are nucleic acid molecules that encode transcriptionand/or translation products that are subsequently spliced to ultimatelyproduce functional PIP-72 polypeptides. Splicing can be accomplished invitro or in vivo, and can involve cis- or trans-splicing. The substratefor splicing can be polynucleotides (e.g., RNA transcripts) orpolypeptides. An example of cis-splicing of a polynucleotide is where anintron inserted into a coding sequence is removed and the two flankingexon regions are spliced to generate a PIP-72 polypeptide encodingsequence. An example of trans splicing would be where a polynucleotideis encrypted by separating the coding sequence into two or morefragments that can be separately transcribed and then spliced to formthe full-length pesticidal encoding sequence. The use of a splicingenhancer sequence, which can be introduced into a construct, canfacilitate splicing either in cis or trans-splicing of polypeptides(U.S. Pat. Nos. 6,365,377 and 6,531,316). Thus, in some embodiments thepolynucleotides do not directly encode a full-length PIP-72 polypeptide,but rather encode a fragment or fragments of a PIP-72 polypeptide. Thesepolynucleotides can be used to express a functional PIP-72 polypeptidethrough a mechanism involving splicing, where splicing can occur at thelevel of polynucleotide (e.g., intron/exon) and/or polypeptide (e.g.,intein/extein). This can be useful, for example, in controllingexpression of pesticidal activity, since a functional pesticidalpolypeptide will only be expressed if all required fragments areexpressed in an environment that permits splicing processes to generatefunctional product. In another example, introduction of one or moreinsertion sequences into a polynucleotide can facilitate recombinationwith a low homology polynucleotide; use of an intron or intein for theinsertion sequence facilitates the removal of the intervening sequence,thereby restoring function of the encoded variant.

Nucleic acid molecules that are fragments of these nucleic acidsequences encoding PIP-72 polypeptides are also encompassed by theembodiments. “Fragment” as used herein refers to a portion of thenucleic acid sequence encoding a PIP-72 polypeptide. A fragment of anucleic acid sequence may encode a biologically active portion of aPIP-72 polypeptide or it may be a fragment that can be used as ahybridization probe or PCR primer using methods disclosed below. Nucleicacid molecules that are fragments of a nucleic acid sequence encoding aPIP-72 polypeptide comprise at least about 130, 140, 150, 160, 170, 180,190, 200, 210, 220, 230, 240, 250 or 260, contiguous nucleotides or upto the number of nucleotides present in a full-length nucleic acidsequence encoding a PIP-72 polypeptide disclosed herein, depending uponthe intended use. “Contiguous nucleotides” is used herein to refer tonucleotide residues that are immediately adjacent to one another.Fragments of the nucleic acid sequences of the embodiments will encodeprotein fragments that retain the biological activity of the PIP-72polypeptide and, hence, retain insecticidal activity. “Retains PIP-72activity” is used herein to refer to a polypeptide having at least about10%, at least about 30%, at least about 50%, at least about 70%, 80%,90%, 95% or higher of the insecticidal activity of the full-lengthPIP-72Aa polypeptide of SEQ ID NO: 2. In one embodiment, theinsecticidal activity is Lepidoptera activity. In one embodiment, theinsecticidal activity is against a Coleopteran species. In oneembodiment, the insecticidal activity is against a Diabrotica species.In one embodiment, the insecticidal activity is against one or moreinsect pests of the corn rootworm complex: Western corn rootworm,Diabrotica virgifera virgifera; northern corn rootworm, D. barberi:Southern corn rootworm or spotted cucumber beetle; Diabroticaundecimpunctata howardi, and the Mexican corn rootworm, D. virgiferazeae. In one embodiment, the insecticidal activity is against Westerncorn rootworm, Diabrotica virgifera virgifera.

In some embodiments a fragment of a nucleic acid sequence encoding aPIP-72 polypeptide encoding a biologically active portion of a proteinwill encode at least about 15, 20, 30, 40, 50, 60, 70, 75, 76, 77, 78,79, 80, 81, 82, 83, 84 or 85, contiguous amino acids or up to the totalnumber of amino acids present in a full-length PIP-72 polypeptide of theembodiments. In some embodiments, the fragment is an N-terminal and/or aC-terminal truncation of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14 or more amino acids from the N-terminus and/or C-terminusrelative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28,SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO:852, any one of SEQ ID NO: 903-914, SEQ ID NO: 927, SEQ ID NO: 928, SEQID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO:936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 or variants thereof, e.g.,by proteolysis, insertion of a start codon, deletion of the codonsencoding the deleted amino acids with the concomitant insertion of astop codon or by insertion of a stop codon in the coding sequence. Insome embodiments, the fragments encompassed herein result from theremoval of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 or14 or more amino acids from the N-terminus relative to SEQ ID NO: 2, SEQID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO:903-914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933,SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ IDNO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945or SEQ ID NO: 946 or variants thereof, e.g., by proteolysis or byinsertion of a start codon in the coding sequence. In some embodiments,the fragments encompassed herein result from the removal of theN-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 amino acidsrelative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28 orSEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO:852, any one of SEQ ID NO: 903-914, SEQ ID NO: 927, SEQ ID NO: 928, SEQID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO:936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946 or variants thereof, e.g.,by proteolysis or by insertion of a start codon in the coding sequence.

In some embodiments a PIP-72 polypeptide is encoded by a nucleic acidsequence sufficiently homologous to the nucleic acid sequence of SEQ IDNO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ IDNO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 27, SEQ ID NO: 31, SEQID NO: 949, SEQ ID NO: 950, SEQ ID NO: 954, SEQ ID NO: 955, SEQ ID NO:956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967 or SEQ ID NO:968. “Sufficiently homologous” is used herein to refer to an amino acidor nucleic acid sequence that has at least about 50%, 55%, 60%, 65%,70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater sequence homologycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding homology of proteins encoded by two nucleic acid sequencesby taking into account codon degeneracy, amino acid similarity, readingframe positioning, and the like. In some embodiments the sequencehomology is against the full length sequence of the polynucleotideencoding a PIP-72 polypeptide or against the full length sequence of aPIP-72 polypeptide. In some embodiments the PIP-72 polypeptide has atleast about 50%, 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity compared to SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 18, SEQ ID NO: 28 or SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928,SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ IDNO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943,SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946. In some embodimentsthe sequence identity is against the full length sequence of thepolynucleotide encoding a PIP-72 polypeptide or against the full lengthsequence of a PIP-72 polypeptide. In some embodiments the sequenceidentity is calculated using ClustalW algorithm in the ALIGNX® module ofthe Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters. In some embodiments the sequence identityis across the entire length of polypeptide calculated using ClustalWalgorithm in the ALIGNX module of the Vector NTI Program Suite(Invitrogen Corporation, Carlsbad, Calif.) with all default parameters.

To determine the percent identity of two amino acid sequences or of twonucleic acid sequences, the sequences are aligned for optimal comparisonpurposes. The percent identity between the two sequences is a functionof the number of identical positions shared by the sequences (i.e.,percent identity=number of identical positions/total number of positions(e.g., overlapping positions)×100). In one embodiment, the two sequencesare the same length. In another embodiment, the comparison is across theentirety of the reference sequence (e.g., across the entirety of one ofSEQ ID NO: 1, SEQ ID NO: 2). The percent identity between two sequencescan be determined using techniques similar to those described below,with or without allowing gaps. In calculating percent identity,typically exact matches are counted.

The determination of percent identity between two sequences can beaccomplished using a mathematical algorithm. A non-limiting example of amathematical algorithm utilized for the comparison of two sequences isthe algorithm of Karlin and Altschul, (1990) Proc. Natl. Acad. Sci. USA87:2264, modified as in Karlin and Altschul, (1993) Proc. Natl. Acad.Sci. USA 90:5873-5877. Such an algorithm is incorporated into the BLASTNand BLASTX programs of Altschul, et al., (1990) J. Mol. Biol. 215:403.BLAST nucleotide searches can be performed with the BLASTN program,score=100, wordlength=12, to obtain nucleic acid sequences homologous topesticidal nucleic acid molecules of the embodiments. BLAST proteinsearches can be performed with the BLASTX program, score=50,wordlength=3, to obtain amino acid sequences homologous to pesticidalprotein molecules of the embodiments. To obtain gapped alignments forcomparison purposes, Gapped BLAST (in BLAST 2.0) can be utilized asdescribed in Altschul, et al., (1997) Nucleic Acids Res. 25:3389.Alternatively, PSI-Blast can be used to perform an iterated search thatdetects distant relationships between molecules. See, Altschul, et al.,(1997) supra. When utilizing BLAST, Gapped BLAST, and PSI-Blastprograms, the default parameters of the respective programs (e.g.,BLASTX and BLASTN) can be used. Alignment may also be performed manuallyby inspection.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the ClustalW algorithm (Higgins, et al.,(1994) Nucleic Acids Res. 22:4673-4680). ClustalW compares sequences andaligns the entirety of the amino acid or DNA sequence, and thus canprovide data about the sequence conservation of the entire amino acidsequence. The ClustalW algorithm is used in several commerciallyavailable DNA/amino acid analysis software packages, such as the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.). After alignment of amino acid sequences withClustalW, the percent amino acid identity can be assessed. Anon-limiting example of a software program useful for analysis ofClustalW alignments is GENEDOC™. GENEDOC™ (Karl Nicholas) allowsassessment of amino acid (or DNA) similarity and identity betweenmultiple proteins. Another non-limiting example of a mathematicalalgorithm utilized for the comparison of sequences is the algorithm ofMyers and Miller, (1988) CABIOS 4:11-17. Such an algorithm isincorporated into the ALIGN program (version 2.0), which is part of theGCG Wisconsin Genetics Software Package, Version 10 (available fromAccelrys, Inc., 9685 Scranton Rd., San Diego, Calif., USA). Whenutilizing the ALIGN program for comparing amino acid sequences, a PAM120weight residue table, a gap length penalty of 12, and a gap penalty of 4can be used.

Another non-limiting example of a mathematical algorithm utilized forthe comparison of sequences is the algorithm of Needleman and Wunsch,(1970) J. Mol. Biol. 48(3):443-453, used GAP Version 10 software todetermine sequence identity or similarity using the following defaultparameters: % identity and % similarity for a nucleic acid sequenceusing GAP Weight of 50 and Length Weight of 3, and the nwsgapdna.cmpiiscoring matrix; % identity or % similarity for an amino acid sequenceusing GAP weight of 8 and length weight of 2, and the BLOSUM62 scoringprogram. Equivalent programs may also be used. “Equivalent program” isused herein to refer to any sequence comparison program that, for anytwo sequences in question, generates an alignment having identicalnucleotide residue matches and an identical percent sequence identitywhen compared to the corresponding alignment generated by GAP Version10.

The embodiments also encompass nucleic acid molecules encoding PIP-72polypeptide variants. “Variants” of the PIP-72 polypeptide encodingnucleic acid sequences include those sequences that encode the PIP-72polypeptides disclosed herein but that differ conservatively because ofthe degeneracy of the genetic code as well as those that aresufficiently identical as discussed above. Naturally occurring allelicvariants can be identified with the use of well-known molecular biologytechniques, such as polymerase chain reaction (PCR) and hybridizationtechniques as outlined below. Variant nucleic acid sequences alsoinclude synthetically derived nucleic acid sequences that have beengenerated, for example, by using site-directed mutagenesis but whichstill encode the PIP-72 polypeptides disclosed as discussed below.

The present disclosure provides isolated or recombinant polynucleotidesthat encode any of the PIP-72 polypeptides disclosed herein. Thosehaving ordinary skill in the art will readily appreciate that due to thedegeneracy of the genetic code, a multitude of nucleotide sequencesencoding PIP-72 polypeptides of the present disclosure exist. Table 1 isa codon table that provides the synonymous codons for each amino acid.For example, the codons AGA, AGG, CGA, CGC, CGG, and CGU all encode theamino acid arginine. Thus, at every position in the nucleic acids of thedisclosure where an arginine is specified by a codon, the codon can bealtered to any of the corresponding codons described above withoutaltering the encoded polypeptide. It is understood that U in an RNAsequence corresponds to T in a DNA sequence.

TABLE 1 Alanine Ala GCA GCC GCG GCU Cysteine Cys UGC UGU  Aspartic acidAsp GAC GAU  Glutamic acid Glu GAA GAG  Phenylalanine Phe UUC UUU Glycine Gly GGA GGC GGG GGU Histidine His CAC CAU  Isoleucine IleAUA AUC AUU Lysine Lys AAA AAG  Leucine Leu UUA UUG CUA CUC CUG CUUMethionine Met AUG  Asparagine Asn AAC AAU  Proline Pro CCA CCC CCG CCUGlutamine Gln CAA CAG  Arginine Arg AGA AGG CGA CGC CGG CGU Serine SerAGC AGU UCA UCC UCG UCU Threonine Thr ACA ACC ACG ACU  Valine ValGUA GUC GUG UU  Tryptophan Trp UGG  Tyrosine Tyr UAC UAU 

The skilled artisan will further appreciate that changes can beintroduced by mutation of the nucleic acid sequences thereby leading tochanges in the amino acid sequence of the encoded PIP-72 polypeptides,without altering the biological activity of the proteins. Thus, variantnucleic acid molecules can be created by introducing one or morenucleotide substitutions, additions and/or deletions into thecorresponding nucleic acid sequence disclosed herein, such that one ormore amino acid substitutions, additions or deletions are introducedinto the encoded protein. Mutations can be introduced by standardtechniques, such as site-directed mutagenesis and PCR-mediatedmutagenesis. Such variant nucleic acid sequences are also encompassed bythe present disclosure.

Alternatively, variant nucleic acid sequences can be made by introducingmutations randomly along all or part of the coding sequence, such as bysaturation mutagenesis, and the resultant mutants can be screened forability to confer pesticidal activity to identify mutants that retainactivity. Following mutagenesis, the encoded protein can be expressedrecombinantly, and the activity of the protein can be determined usingstandard assay techniques.

The polynucleotides of the disclosure and fragments thereof areoptionally used as substrates for a variety of recombination andrecursive recombination reactions, in addition to standard cloningmethods as set forth in, e.g., Ausubel, Berger and Sambrook, i.e., toproduce additional pesticidal polypeptide homologues and fragmentsthereof with desired properties. A variety of such reactions are known,including those developed by the inventors and their co-workers. Methodsfor producing a variant of any nucleic acid listed herein comprisingrecursively recombining such polynucleotide with a second (or more)polynucleotide, thus forming a library of variant polynucleotides arealso embodiments of the disclosure, as are the libraries produced, thecells comprising the libraries and any recombinant polynucleotideproduces by such methods. Additionally, such methods optionally compriseselecting a variant polynucleotide from such libraries based onpesticidal activity, as is wherein such recursive recombination is donein vitro or in vivo.

A variety of diversity generating protocols, including nucleic acidrecursive recombination protocols are available and fully described inthe art. The procedures can be used separately, and/or in combination toproduce one or more variants of a nucleic acid or set of nucleic acids,as well as variants of encoded proteins. Individually and collectively,these procedures provide robust, widely applicable ways of generatingdiversified nucleic acids and sets of nucleic acids (including, e.g.,nucleic acid libraries) useful, e.g., for the engineering or rapidevolution of nucleic acids, proteins, pathways, cells and/or organismswith new and/or improved characteristics.

While distinctions and classifications are made in the course of theensuing discussion for clarity, it will be appreciated that thetechniques are often not mutually exclusive. Indeed, the various methodscan be used singly or in combination, in parallel or in series, toaccess diverse sequence variants.

The result of any of the diversity generating procedures describedherein can be the generation of one or more nucleic acids, which can beselected or screened for nucleic acids with or which confer desirableproperties or that encode proteins with or which confer desirableproperties. Following diversification by one or more of the methodsherein or otherwise available to one of skill, any nucleic acids thatare produced can be selected for a desired activity or property, e.g.pesticidal activity or, such activity at a desired pH, etc. This caninclude identifying any activity that can be detected, for example, inan automated or automatable format, by any of the assays in the art,see, e.g., discussion of screening of insecticidal activity, infra. Avariety of related (or even unrelated) properties can be evaluated, inserial or in parallel, at the discretion of the practitioner.

Descriptions of a variety of diversity generating procedures forgenerating modified nucleic acid sequences, e.g., those coding forpolypeptides having pesticidal activity or fragments thereof, are foundin the following publications and the references cited therein: Soong,et al., (2000) Nat Genet 25(4):436-439; Stemmer, et al., (1999) TumorTargeting 4:1-4; Ness, et al., (1999) Nat Biotechnol 17:893-896; Chang,et al., (1999) Nat Biotechnol 17:793-797; Minshull and Stemmer, (1999)Curr Opin Chem Biol 3:284-290; Christians, et al., (1999) Nat Biotechnol17:259-264; Crameri, et al., (1998) Nature 391:288-291; Crameri, et al.,(1997) Nat Biotechnol 15:436-438; Zhang, et al., (1997) PNAS USA94:4504-4509; Patten, et al., (1997) Curr Opin Biotechnol 8:724-733;Crameri, et al., (1996) Nat Med 2:100-103; Crameri, et al., (1996) NatBiotechnol 14:315-319; Gates, et al., (1996) J Mol Biol 255:373-386;Stemmer, (1996) “Sexual PCR and Assembly PCR” In: The Encyclopedia ofMolecular Biology. VCH Publishers, New York. pp. 447-457; Crameri andStemmer, (1995) BioTechniques 18:194-195; Stemmer, et al., (1995) Gene,164:49-53; Stemmer, (1995) Science 270: 1510; Stemmer, (1995)Bio/Technology 13:549-553; Stemmer, (1994) Nature 370:389-391 andStemmer, (1994) PNAS USA 91:10747-10751.

Mutational methods of generating diversity include, for example,site-directed mutagenesis (Ling, et al., (1997) Anal Biochem254(2):157-178; Dale, et al., (1996) Methods Mol Biol 57:369-374; Smith,(1985) Ann Rev Genet 19:423-462; Botstein and Shortle, (1985) Science229:1193-1201; Carter, (1986) Biochem J 237:1-7 and Kunkel, (1987) “Theefficiency of oligonucleotide directed mutagenesis” in Nucleic Acids &Molecular Biology (Eckstein and Lilley, eds., Springer Verlag, Berlin));mutagenesis using uracil containing templates (Kunkel, (1985) PNAS USA82:488-492; Kunkel, et al., (1987) Methods Enzymol 154:367-382 and Bass,et al., (1988) Science 242:240-245); oligonucleotide-directedmutagenesis (Zoller and Smith, (1983) Methods Enzymol 100:468-500;Zoller and Smith, (1987) Methods Enzymol 154:329-350 (1987); Zoller andSmith, (1982) Nucleic Acids Res 10:6487-6500), phosphorothioate-modifiedDNA mutagenesis (Taylor, et al., (1985) Nucl Acids Res 13:8749-8764;Taylor, et al., (1985) Nucl Acids Res 13:8765-8787 (1985); Nakamaye andEckstein, (1986) Nucl Acids Res 14:9679-9698; Sayers, et al., (1988)Nucl Acids Res 16:791-802 and Sayers, et al., (1988) Nucl Acids Res16:803-814); mutagenesis using gapped duplex DNA (Kramer, et al., (1984)Nucl Acids Res 12:9441-9456; Kramer and Fritz, (1987) Methods Enzymol154:350-367; Kramer, et al., (1988) Nucl Acids Res 16:7207 and Fritz, etal., (1988) Nucl Acids Res 16:6987-6999).

Additional suitable methods include point mismatch repair (Kramer, etal., (1984) Cell 38:879-887), mutagenesis using repair-deficient hoststrains (Carter, et al., (1985) Nucl Acids Res 13:4431-4443 and Carter,(1987) Methods in Enzymol 154:382-403), deletion mutagenesis(Eghtedarzadeh and Henikoff, (1986) Nucl Acids Res 14:5115),restriction-selection and restriction-purification (Wells, et al.,(1986) Phil Trans R Soc Lond A 317:415-423), mutagenesis by total genesynthesis (Nambiar, et al., (1984) Science 223:1299-1301; Sakamar andKhorana, (1988) Nucl Acids Res 14:6361-6372; Wells, et al., (1985) Gene34:315-323 and Grundström, et al., (1985) Nucl Acids Res 13:3305-3316),double-strand break repair (Mandecki, (1986) PNAS USA, 83:7177-7181 andArnold, (1993) Curr Opin Biotech 4:450-455). Additional details on manyof the above methods can be found in Methods Enzymol Volume 154, whichalso describes useful controls for trouble-shooting problems withvarious mutagenesis methods.

Additional details regarding various diversity generating methods can befound in the following US Patents, PCT Publications and Applications andEPO publications: U.S. Pat. Nos. 5,723,323, 5,763,192, 5,814,476,5,817,483, 5,824,514, 5,976,862, 5,605,793, 5,811,238, 5,830,721,5,834,252, 5,837,458, WO 1995/22625, WO 1996/33207, WO 1997/20078, WO1997/35966, WO 1999/41402, WO 1999/41383, WO 1999/41369, WO 1999/41368,EP 752008, EP 0932670, WO 1999/23107, WO 1999/21979, WO 1998/31837, WO1998/27230, WO 1998/27230, WO 2000/00632, WO 2000/09679, WO 1998/42832,WO 1999/29902, WO 1998/41653, WO 1998/41622, WO 1998/42727, WO2000/18906, WO 2000/04190, WO 2000/42561, WO 2000/42559, WO 2000/42560,WO 2001/23401 and PCT/US01/06775.

The nucleotide sequences of the embodiments can also be used to isolatecorresponding sequences from other organisms, particularly otherbacteria, particularly a Pseudomonas species and more particularly aPseudomonas putida, a Pseudomonas fulva or a Pseudomonas chlororaphisstrain. In this manner, methods such as PCR, hybridization, and the likecan be used to identify such sequences based on their sequence homologyto the sequences set forth herein. Sequences that are selected based ontheir sequence identity to the entire sequences set forth herein or tofragments thereof are encompassed by the embodiments. Such sequencesinclude sequences that are orthologs of the disclosed sequences. Theterm “orthologs” refers to genes derived from a common ancestral geneand which are found in different species as a result of speciation.Genes found in different species are considered orthologs when theirnucleotide sequences and/or their encoded protein sequences sharesubstantial identity as defined elsewhere herein. Functions of orthologsare often highly conserved among species.

In a PCR approach, oligonucleotide primers can be designed for use inPCR reactions to amplify corresponding DNA sequences from cDNA orgenomic DNA extracted from any organism of interest. Methods fordesigning PCR primers and PCR cloning are generally known in the art andare disclosed in Sambrook, et al., (1989) Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press,Plainview, N.Y.), hereinafter “Sambrook”. See also, Innis, et al., eds.(1990) PCR Protocols: A Guide to Methods and Applications (AcademicPress, New York); Innis and Gelfand, eds. (1995) PCR Strategies(Academic Press, New York); and Innis and Gelfand, eds. (1999) PCRMethods Manual (Academic Press, New York). Known methods of PCR include,but are not limited to, methods using paired primers, nested primers,single specific primers, degenerate primers, gene-specific primers,vector-specific primers, partially-mismatched primers, and the like.

To identify potential PIP-72 polypeptides from bacterial collections,the bacterial cell lysates can be screened with antibodies generatedagainst a PIP-72 polypeptide using Western blotting and/or ELISAmethods. This type of assays can be performed in a high throughputfashion. Positive samples can be further analyzed by various techniquessuch as antibody based protein purification and identification. Methodsof generating antibodies are well known in the art as discussed infra.

Alternatively, mass spectrometry based protein identification method canbe used to identify homologs of PIP-72 polypeptides using protocols inthe literatures (Scott Patterson, (1998), 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Specifically,LC-MS/MS based protein identification method is used to associate the MSdata of given cell lysate or desired molecular weight enriched samples(excised from SDS-PAGE gel of relevant molecular weight bands to PIP-72)with sequence information of PIP-72 (e.g., SEQ ID NO: 2)) and itshomologs. Any match in peptide sequences indicates the potential ofhaving the homologs in the samples. Additional techniques (proteinpurification and molecular biology) can be used to isolate the proteinand identify the sequences of the homologs.

In hybridization methods, all or part of the pesticidal nucleic acidsequence can be used to screen cDNA or genomic libraries. Methods forconstruction of such cDNA and genomic libraries are generally known inthe art and are disclosed in Sambrook and Russell, (2001), supra. Theso-called hybridization probes may be genomic DNA fragments, cDNAfragments, RNA fragments or other oligonucleotides and may be labeledwith a detectable group such as 32P or any other detectable marker, suchas other radioisotopes, a fluorescent compound, an enzyme or an enzymeco-factor. Probes for hybridization can be made by labeling syntheticoligonucleotides based on the known PIP-72 polypeptide-encoding nucleicacid sequence disclosed herein. Degenerate primers designed on the basisof conserved nucleotides or amino acid residues in the nucleic acidsequence or encoded amino acid sequence can additionally be used. Theprobe typically comprises a region of nucleic acid sequence thathybridizes under stringent conditions to at least about 12, at leastabout 25, at least about 50, 75, 100, 125, 150, 175 or 200 consecutivenucleotides of nucleic acid sequence encoding a PIP-72 polypeptide ofthe disclosure or a fragment or variant thereof. Methods for thepreparation of probes for hybridization are generally known in the artand are disclosed in Sambrook and Russell, (2001), supra, hereinincorporated by reference.

For example, an entire nucleic acid sequence, encoding a PIP-72polypeptide, disclosed herein or one or more portions thereof may beused as a probe capable of specifically hybridizing to correspondingnucleic acid sequences encoding PIP-72 polypeptide-like sequences andmessenger RNAs. To achieve specific hybridization under a variety ofconditions, such probes include sequences that are unique and arepreferably at least about 10 nucleotides in length or at least about 20nucleotides in length. Such probes may be used to amplify correspondingpesticidal sequences from a chosen organism by PCR. This technique maybe used to isolate additional coding sequences from a desired organismor as a diagnostic assay to determine the presence of coding sequencesin an organism. Hybridization techniques include hybridization screeningof plated DNA libraries (either plaques or colonies; see, for example,Sambrook, et al., (1989) Molecular Cloning: A Laboratory Manual (2d ed.,Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

Hybridization of such sequences may be carried out under stringentconditions. “Stringent conditions” or “stringent hybridizationconditions” is used herein to refer to conditions under which a probewill hybridize to its target sequence to a detectably greater degreethan to other sequences (e.g., at least 2-fold over background).Stringent conditions are sequence-dependent and will be different indifferent circumstances. By controlling the stringency of thehybridization and/or washing conditions, target sequences that are 100%complementary to the probe can be identified (homologous probing).Alternatively, stringency conditions can be adjusted to allow somemismatching in sequences so that lower degrees of similarity aredetected (heterologous probing). Generally, a probe is less than about1000 nucleotides in length, preferably less than 500 nucleotides inlength.

Typically, stringent conditions will be those in which the saltconcentration is less than about 1.5 M Na ion, typically about 0.01 to1.0 M Na ion concentration (or other salts) at pH 7.0 to 8.3 and thetemperature is at least about 30° C. for short probes (e.g., 10 to 50nucleotides) and at least about 60° C. for long probes (e.g., greaterthan 50 nucleotides). Stringent conditions may also be achieved with theaddition of destabilizing agents such as formamide. Exemplary lowstringency conditions include hybridization with a buffer solution of 30to 35% formamide, 1 M NaCl, 1% SDS (sodium dodecyl sulphate) at 37° C.,and a wash in 1× to 2×SSC (20×SSC=3.0 M NaCl/0.3 M trisodium citrate) at50 to 55° C. Exemplary moderate stringency conditions includehybridization in 40 to 45% formamide, 1.0 M NaCl, 1% SDS at 37° C., anda wash in 0.5× to 1×SSC at 55 to 60° C. Exemplary high stringencyconditions include hybridization in 50% formamide, 1 M NaCl, 1% SDS at37° C., and a wash in 0.1×SSC at 60 to 65° C. Optionally, wash buffersmay comprise about 0.1% to about 1% SDS. Duration of hybridization isgenerally less than about 24 hours, usually about 4 to about 12 hours.

Specificity is typically the function of post-hybridization washes, thecritical factors being the ionic strength and temperature of the finalwash solution. For DNA-DNA hybrids, the Tm can be approximated from theequation of Meinkoth and Wahl, (1984) Anal. Biochem. 138:267-284:Tm=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61 (% form)−500/L; where M is themolarity of monovalent cations, % GC is the percentage of guanosine andcytosine nucleotides in the DNA, % form is the percentage of formamidein the hybridization solution, and L is the length of the hybrid in basepairs. The Tm is the temperature (under defined ionic strength and pH)at which 50% of a complementary target sequence hybridizes to aperfectly matched probe. Tm is reduced by about 1° C. for each 1% ofmismatching; thus, Tm, hybridization, and/or wash conditions can beadjusted to hybridize to sequences of the desired identity. For example,if sequences with 90% identity are sought, the Tm can be decreased 10°C. Generally, stringent conditions are selected to be about 5° C. lowerthan the thermal melting point (Tm) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3or 4° C. lower than the thermal melting point (Tm); moderately stringentconditions can utilize a hybridization and/or wash at 6, 7, 8, 9 or 10°C. lower than the thermal melting point (Tm); low stringency conditionscan utilize a hybridization and/or wash at 11, 12, 13, 14, 15 or 20° C.lower than the thermal melting point (Tm). Using the equation,hybridization and wash compositions, and desired Tm, those of ordinaryskill will understand that variations in the stringency of hybridizationand/or wash solutions are inherently described. If the desired degree ofmismatching results in a Tm of less than 45° C. (aqueous solution) or32° C. (formamide solution), it is preferred to increase the SSCconcentration so that a higher temperature can be used. An extensiveguide to the hybridization of nucleic acids is found in Tijssen, (1993)Laboratory Techniques in Biochemistry and MolecularBiology-Hybridization with Nucleic Acid Probes, Part I, Chapter 2(Elsevier, N.Y.); and Ausubel, et al., eds. (1995) Current Protocols inMolecular Biology, Chapter 2 (Greene Publishing and Wiley-Interscience,New York). See, Sambrook, et al., (1989) Molecular Cloning: A LaboratoryManual (2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor,N.Y.).

In some embodiments nucleic acid molecules are provided that encode apolypeptide comprising an amino acid sequence having at least 75%, atleast 80%, at least 85%, at least 90%, at least 95% or greater sequenceidentity to the amino acid sequence set forth in SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ I NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO:937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQ ID NO: 948wherein the polypeptide has insecticidal activity.

Proteins and Variants and Fragments Thereof

PIP-72 polypeptides are also encompassed by the disclosure. “PseudomonasInsecticidal Protein-72”, “PIP-72 polypeptide” or “PIP-72 protein” asused herein interchangeably refers to a polypeptide having pesticidalactivity including but not limited to insecticidal activity against oneor more insect pests of the Coleoptera order, and is sufficientlyhomologous to the protein of SEQ ID NO: 2. A variety of PIP-72polypeptides are contemplated. Sources of polynucleotides that encodePIP-72 polypeptides or related proteins include but are not limited to:a Pseudomonas chlororaphis strain which contains the PIP-72Aapolynucleotide of SEQ ID NO: 1 encoding the PIP-72Aa polypeptide of SEQID NO: 2; a Pseudomonas rhodesiae strain which contains the PIP-72Bapolynucleotide of SEQ ID NO: 3 encoding the PIP-72Ba polypeptide of SEQID NO: 4; a Pseudomonas chlororaphis strain which contains the PIP-72Capolynucleotide of SEQ ID NO: 5 encoding the PIP-72Ca polypeptide of SEQID NO: 6; a Pseudomonas mandelii strain which contains the PIP-72Cbpolynucleotide of SEQ ID NO: 7 encoding the PIP-72Cb polypeptide of SEQID NO: 8; a Pseudomonas congelans strain which contains the PIP-72 Dapolynucleotide of SEQ ID NO: 9 encoding the PIP-72 Da polypeptide of SEQID NO: 10; a Pseudomonas mandelii strain which contains the PIP-72Dbpolynucleotide of SEQ ID NO: 11 encoding the PIP-72Db polypeptide of SEQID NO: 12; a Pseudomonas ficuserectae strain which contains the PIP-72Dcpolynucleotide of SEQ ID NO: 13 encoding the PIP-72Dc polypeptide of SEQID NO: 14; a Pseudomonas mosselii strain which contains the PIP-72Fapolynucleotide of SEQ ID NO: 17 encoding the PIP-72Fa polypeptide of SEQID NO: 18; a Pseudomonas chlororaphis strain which contains the PIP-72Ffpolynucleotide of SEQ ID NO: 27 encoding the PIP-72Ff polypeptide of SEQID NO: 28 and a Pseudomonas chlororaphis strain which contains thePIP-72Gb polynucleotide of SEQ ID NO: 31 encoding the PIP-72Gbpolypeptide of SEQ ID NO: 32; a Pseudomonas chlororaphis strain whichcontains the PIP-72Ab polynucleotide of SEQ ID NO: 949 encoding thePIP-72Ab polypeptide of SEQ ID NO: 927; a Pseudomonas brassicacearumstrain which contains the PIP-72Bb polynucleotide of SEQ ID NO: 950encoding the PIP-72Ab polypeptide of SEQ ID NO: 928; a Pseudomonasentomophila strain which contains the PIP-72Fh polynucleotide of SEQ IDNO: 954 encoding the PIP-72AFh polypeptide of SEQ ID NO: 932; aPseudomonas entomophila strain which contains the PIP-72Fhpolynucleotide of SEQ ID NO: 955 encoding the PIP-72AFh polypeptide ofSEQ ID NO: 933; a Pseudomonas chlororaphis strain which contains thePIP-72Fj polynucleotide of SEQ ID NO: 956 encoding the PIP-72Fjpolypeptide of SEQ ID NO: 934; a Pseudomonas chlororaphis strain whichcontains the PIP-72Fk polynucleotide of SEQ ID NO: 957 encoding thePIP-72Fk polypeptide of SEQ ID NO: 935; a Burkholderia multivoransstrain which contains the PIP-72FI polynucleotide of SEQ ID NO: 958encoding the PIP-72FI polypeptide of SEQ ID NO: 936; a Pseudomonaschlororaphis strain which contains the PIP-72Gg polynucleotide of SEQ IDNO: 961 encoding the PIP-72Gg polypeptide of SEQ ID NO: 939; aPseudomonas chlororaphis strain which contains the PIP-72Ghpolynucleotide of SEQ ID NO: 962 encoding the PIP-72Gh polypeptide ofSEQ ID NO: 940; a Pseudomonas mosselii strain which contains thePIP-72Gi polynucleotide of SEQ ID NO: 963 encoding the PIP-72Gipolypeptide of SEQ ID NO: 941; a Pseudomonas protegens strain whichcontains the PIP-72Gk polynucleotide of SEQ ID NO: 965 encoding thePIP-72Gk polypeptide of SEQ ID NO: 943; a Pseudomonas plecoglossicidastrain which contains the PIP-72GI polynucleotide of SEQ ID NO: 966encoding the PIP-72GI polypeptide of SEQ ID NO: 944; and a Pseudomonaschlororaphis strain which contains the PIP-72Gn polynucleotide of SEQ IDNO: 968 encoding the PIP-72Gn polypeptide of SEQ ID NO: 946. In someembodiments, the insecticidal activity is against western corn rootworm,Diabrotica virgifera virgifera.

In some embodiments a PIP-72 polypeptide is sufficiently homologous tothe amino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18,SEQ ID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO:932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO:944, SEQ ID NO: 945 or SEQ ID NO: 946. “Sufficiently homologous” is usedherein to refer to an amino acid sequence that has at least about 50%,51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%,78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,93%, 94%, 95%, 96%, 97%, 98% or 99% or greater sequence homologycompared to a reference sequence using one of the alignment programsdescribed herein using standard parameters. One of skill in the art willrecognize that these values can be appropriately adjusted to determinecorresponding homology of proteins taking into account amino acidsimilarity and the like. In some embodiments the sequence homology isagainst the full length sequence a PIP-72 polypeptide. In someembodiments the PIP-72 polypeptide has at least about 50%, 51%, 52%,53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%,67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%,81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98% or 99% or greater sequence identity compared to SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32,SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ IDNO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940,SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ IDNO: 946. In some embodiments the sequence identity is against the fulllength sequence of a PIP-72 polypeptide. In some embodiments thesequence identity is calculated using ClustalW algorithm in the ALIGNX®module of the Vector NTI® Program Suite (Invitrogen Corporation,Carlsbad, Calif.) with all default parameters. In some embodiments thesequence identity is across the entire length of polypeptide calculatedusing ClustalW algorithm in the ALIGNX® module of the Vector NTI®Program Suite (Invitrogen Corporation, Carlsbad, Calif.) with alldefault parameters.

As used herein, the terms “protein,” “peptide molecule,” or“polypeptide” includes any molecule that comprises five or more aminoacids. It is well known in the art that protein, peptide or polypeptidemolecules may undergo modification, including post-translationalmodifications, such as, but not limited to, disulfide bond formation,glycosylation, phosphorylation or oligomerization. Thus, as used herein,the terms “protein,” “peptide molecule” or “polypeptide” includes anyprotein that is modified by any biological or non-biological process.The terms “amino acid” and “amino acids” refer to all naturallyoccurring L-amino acids.

A “recombinant protein” is used herein to refer to a protein that is nolonger in its natural environment, for example in vitro or in arecombinant bacterial or plant host cell. A PIP-72 polypeptide that issubstantially free of cellular material includes preparations of proteinhaving less than about 30%, 20%, 10% or 5% (by dry weight) ofnon-pesticidal protein (also referred to herein as a “contaminatingprotein”).

“Fragments” or “biologically active portions” include polypeptidefragments comprising amino acid sequences sufficiently identical to aPIP-72 polypeptide and that exhibit insecticidal activity. “Fragments”or “biologically active portions” of PIP-72 polypeptides includesfragments comprising amino acid sequences sufficiently identical to theamino acid sequence set forth in SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO:10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ IDNO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO:825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, anyone of SEQ ID NO: 903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928,SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ IDNO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943,SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946, respectively. Abiologically active portion of a PIP-72 polypeptide can be a polypeptidethat is, for example, 10, 25, 50, 55, 60, 61, 62, 63, 64, 65, 66, 67,68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85amino acids in length. Such biologically active portions can be preparedby recombinant techniques and evaluated for insecticidal activity. Asused here, a fragment comprises at least 8 contiguous amino acids of aPIP-72 polypeptide. In some embodiments a PIP-72 polypeptide fragmentcomprises at least 8 contiguous amino acids of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO:528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO:771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903-SEQ IDNO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933,SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ IDNO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945or SEQ ID NO: 946. In some embodiments, the PIP-72 polypeptide fragmentis an N-terminal and/or a C-terminal truncation of at least about 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 or more amino acids from theN-terminus and/or C-terminus relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771,SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903-SEQ ID NO:914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO:940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 orSEQ ID NO: 946, e.g., by proteolysis, by insertion of a start codon, bydeletion of the codons encoding the deleted amino acids and concomitantinsertion of a start codon, and/or insertion of a stop codon.

In some embodiments, the PIP-72 polypeptide fragments encompassed hereinresult from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 or more amino acids relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ IDNO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ IDNO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ IDNO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903-SEQ ID NO: 914,SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ IDNO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940,SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ IDNO: 946, e.g., by proteolysis or by insertion of a start codon, bydeletion of the codons encoding the deleted amino acids and concomitantinsertion of a start codon.

In some embodiments, the PIP-72 polypeptide fragments encompassed hereinresult from the removal of the N-terminal 1, 2, 3, 4, 5, 6, 7, 8, 9 or10 amino acids relative to SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8 or variants thereof including, but not limited to any one ofSEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844,SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO:903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO:939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQID NO: 945 or SEQ ID NO: 946. In some embodiments the truncation is ofthe first 4 amino acids of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18,SEQ ID NO: 28, SEQ ID NO: 32 or variants thereof including, but notlimited to any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ IDNO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852,any one of SEQ ID NO: 903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO:928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO:943, SEQ ID NO: 944, SEQ ID NO: 945 or SEQ ID NO: 946.

“Variants” as used herein refers to proteins or polypeptides having anamino acid sequence that is at least about 50%, 51%, 52%, 53%, 54%, 55%,56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%,84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%,98% or 99% identical to the parental amino acid sequence. The term“about” as used herein with respect to % sequence identity means up toand including ±0.5% in 0.1% increments. For example “about 90%” sequenceidentity includes 89.5%, 89.6%, 89.7%, 89.8%, 89.9%, 90%, 90.1%, 90.2%,90.3%, 90.4% and 90.5%.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% identity across the entire length of theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQID NO: 28, SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO:932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO:944, SEQ ID NO: 945 or SEQ ID NO: 946.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 4.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 6.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 8.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 10.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 12.

In some embodiments a PIP-72 polypeptide has at least about 50%, 51%,52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%,66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 78%,80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98%, 99% or greater identity across the entirelength of the amino acid sequence of SEQ ID NO: 14.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 18.

In some embodiments a PIP-72 polypeptide has at least about 85%, 86%,87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater identity across the entire length of the amino acid sequence ofSEQ ID NO: 28.

In some embodiments a PIP-72 polypeptide has at least about 95%, 96%,97%, 98%, 99% or greater identity across the entire length of the aminoacid sequence of SEQ ID NO: 32.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 927.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 928.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 932.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 933.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 934.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 935.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 936.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 939.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 940.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 941.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 943.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 944.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 945.

In some embodiments a PIP-72 polypeptide has at least about 60%, 61%,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%,76%, 77%, 78%, 78%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%,90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or greater identityacross the entire length of the amino acid sequence of SEQ ID NO: 946.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidalactivity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 70% identity to the amino acid sequence of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12 or SEQ ID NO: 14, wherein the polypeptide has insecticidalactivity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 2, wherein the polypeptide has insecticidal activity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 4, wherein the polypeptide has insecticidal activity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 6, wherein the polypeptide has insecticidal activity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 8, wherein the polypeptide has insecticidal activity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 10, wherein the polypeptide has insecticidal activity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 12, wherein the polypeptide has insecticidal activity.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence having at least 50% identity to the amino acid sequence of SEQID NO: 14, wherein the polypeptide has insecticidal activity.

In some embodiments the sequence identity is across the entire length ofpolypeptide calculated using ClustalW algorithm in the ALIGNX® module ofthe Vector NTI® Program Suite (Invitrogen Corporation, Carlsbad, Calif.)with all default parameters.

In some embodiments the PIP-72 polypeptide comprises an amino acid motifas represented by amino acid residues 37-51 of SEQ ID NO: 846, SEQ IDNO: 847, SEQ ID NO: 848 or SEQ ID NO: 849.

In some embodiments, the PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 2 having an amino acid substitution at one ormore residues selected from residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70,71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, of SEQID NO: 2 in any combination, and optionally the PIP-72 polypeptidefurther comprises a deletion of 1 to 5 amino acids, an insertion of 1 to5 amino acids, addition of one or more amino acids at the N-terminusand/or addition of one or more amino acids at the C-terminus relative toSEQ ID NO: 2, in any combination.

In some embodiments, the PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 2 having an amino acid substitution at one ormore residues selected from residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12,13, 14, 15, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 56, 58, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86 of SEQ ID NO: 2, in anycombination, and optionally the PIP-72 polypeptide further comprises adeletion of 1 to 5 amino acids, an insertion of 1 to 5 amino acids,addition of one or more amino acids at the N-terminus or addition of oneor more amino acids at the C-terminus relative to SEQ ID NO: 2, in anycombination.

In some embodiments, the PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 2 having an amino acid substitution at 1 to 45residues selected from residues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51,52, 53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 and 86, of SEQ IDNO: 2 in any combination, and optionally the PIP-72 polypeptide furthercomprises a deletion of 1 to 5 amino acids, an insertion of 1 to 5 aminoacids, addition of one or more amino acids at the N-terminus and/oraddition of one or more amino acids at the C-terminus relative to SEQ IDNO: 2, in any combination.

In some embodiments, the PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 2 having an amino acid substitution compared tothe native amino acid of SEQ ID NO: 2 at 1 to 45 residues selected fromresidues 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 17, 18, 19, 20,22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39,40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 58, 60, 63, 64,65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85 and 86 of SEQ ID NO: 2, in any combination, and optionallythe PIP-72 polypeptide further comprises a deletion of 1 to 5 aminoacids, an insertion of 1 to 5 amino acids, addition of one or more aminoacids at the N-terminus and/or addition of one or more amino acids atthe C-terminus relative to SEQ ID NO: 2, in any combination.

In specific embodiments, the substitution is an alanine for the nativeamino acid at the recited position(s). Also encompassed are the nucleicacid sequence(s) encoding the variant protein or polypeptide.

In some embodiments the PIP-72 polypeptide comprising an amino acidsequence of SEQ ID NO: 846, wherein Xaa at position 2 is Gly, Ala, Cys,Asp, Glu, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, Trp or Tyr; Xaa atposition 3 is Ile or Trp; Xaa at position 4 is Thr, Ala, Asp, Glu, His,Ile, Lys, Leu, Arg, Ser, Val, Trp or Tyr; Xaa at position 5 is Val, Ala,Cys, Gly, His, Ile or Tyr; Xaa at position 6 is Thr, Ala, Cys, Phe, Gly,His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Trp or Tyr; Xaa at position 7 isAsn, Ala or Val; Xaa at position 8 is Asn, Ala, Cys, Asp, Glu, Gly, His,Ile, Lys, Leu, Met, Gln, Arg, Ser, Thr or Val; Xaa at position 9 is Ser,Ala, Cys, Gly or Thr; Xaa at position 10 is Ser, Ala, Glu, Phe, Gly,His, Ile, Lys, Leu, Asn, Pro, Gln, Arg, Thr or Trp; Xaa at position 11is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Ser, Thr,Val or Tyr; Xaa at position 12 is Pro, Ala, Cys, Asp, Glu, Gly, His,Lys, Leu, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 13is Ile, Asn, Gln or Val; Xaa at position 14 is Glu, Ala, Cys, Phe, His,Lys or Gln; Xaa at position 15 is Val, Ala, Cys, Ile, Met or Arg; Xaa atposition 17 is Ile, Glu or Val; Xaa at position 18 is Asn or Ser; Xaa atposition 19 is His, Ala, Glu, Lys, Leu, Pro, Arg, Ser or Tyr; Xaa atposition 20 is Trp, Ala or Thr; Xaa at position 22 is Ser, Ala, Asp,Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val or Tyr;Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thror Val; Xaa at position 24 is Gly, Asp or Phe; Xaa at position 25 isAsp, Ala, Glu, Phe, Asn or Gln; Xaa at position 26 is Thr, Glu or Pro;Xaa at position 27 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, Gln,Arg or Thr; Xaa at position 28 is Phe, Pro, Trp or Tyr; Xaa at position29 is Phe, Ala, Cys, Ile, Leu, Gln, Arg, Trp or Tyr; Xaa at position 30is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln,Arg, Thr, Val, Trp or Tyr; Xaa at position 31 is Val, Ile or Leu; Xaa atposition 32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 33 is Asn, Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr,Val or Tyr; Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met,Asn, Gln, Arg, Ser, Thr or Tyr; Xaa at position 35 is Lys, Ala, Cys,Asp, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa atposition 36 is Gln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro,Arg, Ser, Thr or Val; Xaa at position 37 is Glu, Ala, Cys, Asp, Phe,Gly, Ile, Lys, Leu, Met, Asn, Ser, Thr or Val; Xaa at position 38 isThr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg,Ser, Val, Trp or Tyr; Xaa at position 39 is Trp or Phe; Xaa at position40 is Asp, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 42 is Ser, Ala, Cys,Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp orTyr; Xaa at position 44 is Ser, Ala, Asp, Glu, Gly, Leu, Met, Asn, Pro,Gln, Thr, Val or Tyr; Xaa at position 45 is Arg, Lys or Ser; Xaa atposition 46 is Gly, Ala or Gln; Xaa at position 47 is Phe, Cys, Val orTyr; Xaa at position 48 is Val, Ile or Leu; Xaa at position 49 is Leu,Cys, Phe, Met, Arg or Tyr; Xaa at position 50 is Ser, Ala, Cys, Asp,Ile, Met, Pro, Gln, Thr or Val; Xaa at position 51 is Leu, Ala, Cys, Metor Val; Xaa at position 52 is Lys, Cys, Phe, His, Ile, Leu, Met, Asn,Arg, Ser, Thr, Trp or Tyr; Xaa at position 53 is Lys, Ala, Cys, Asp,Glu, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val or Tyr; Xaaat position 54 is Asn, Cys, Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Seror Trp; Xaa at position 56 is Ala, Gly, Leu, Asn, Pro, Gln, Arg, Ser orThr; Xaa at position 57 is Gln, Glu, Leu, Met, Ser or Thr; Xaa atposition 58 is His, Ala, Asp, Phe, Leu, Met, Asn, Arg, Trp or Tyr; Xaaat position 60 is Tyr, Glu or Phe; Xaa at position 63 is Gln, Cys, Gly,Ile, Leu, Met, Asn, Thr, Val or Tyr; Xaa at position 64 is Ala, Phe,Gly, His, Arg, Ser or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Leu, Asn, Thr or Val; Xaa at position 66 isSer, Ala or Gly; Xaa at position 67 is Lys, Ala, Cys, Asp, Phe, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position68 is Ile Asp, Leu or Val; Xaa at position 69 is Glu, Ala, Cys, Asp,Phe, His, Ile, Leu, Met, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position70 is Val, Cys or Ile; Xaa at position 71 is Asp, Ala, Cys, Gly, His,Ile, Leu, Met, Asn, Ser, Thr, Val or Tyr; Xaa at position 72 is Asn,Ala, Cys, Asp, Glu, Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val or Trp;Xaa at position 73 is Asn, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Ser,Thr, Val or Tyr; Xaa at position 74 is Ala, Cys, Asp, Phe, Gly, His,Ile, Leu, Asn, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 75 isVal, Cys, Ile or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His,Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 77 is AspTyr; Xaa at position 78 is Gln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu,Met, Asn, Arg, Ser, Thr, Val or Tyr; Xaa at position 79 is Gly, Arg,Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp orTyr; Xaa at position 80 is Arg, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu,Asn, Ser, Thr, Val or Tyr; Xaa at position 81 is Leu, Ala, Cys, Asp,Phe, Gly, His, Ile, Asn, Pro, Arg, Ser, Thr or Val; Xaa at position 82is Ile, Ala, Leu, Met, Arg or Val; Xaa at position 83 is Glu, Ala, Cys,Asp, Phe, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr, Val or Tyr;Xaa at position 84 is Pro, Ala, Cys, Glu, Ile, Ser, Val, Trp or Tyr; Xaaat position 85 is Leu, Cys, Gly or Val; and Xaa at position 86 is Ser,Ala, Ile, Thr or Val, and wherein 1 to 14 amino acids are optionallydeleted from the N-terminus and/or C-terminus of the PIP-72 polypeptide.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 846 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45, amino acidsubstitutions, in any combination, at residues designated by Xaa in SEQID NO: 846 compared to the native amino acid at the correspondingposition of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 846 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29amino acid substitutions, in any combination, at residues designated byXaa in SEQ ID NO: 846 compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 847, wherein Xaa at position 2 is Gly, Lys orAla; Xaa at position 3 is Ile or Leu; Xaa at position 4 is Thr or Ser;Xaa at position 5 is Val or Ile; Xaa at position 6 is Thr or Lys; Xaa atposition 8 is Asn, Lys, Gly or Ser; Xaa at position 9 is Ser or Ala; Xaaat position 11 is Asn, Lys, His or Thr; Xaa at position 12 is Pro, Thr,Lys or Ser; Xaa at position 13 is Ile or Val; Xaa at position 14 is Gluor Asp; Xaa at position 15 is Val, Ala or Ile; Xaa at position 16 is Alaor Ser; Xaa at position 17 is Ile or Val; Xaa at position 18 is Asn orSer; Xaa at position 19 is His, Lys, Arg, Gln or Ala; Xaa at position 21is Gly or Arg; Xaa at position 22 is Ser, Lys, Asn, Asp or Thr; Xaa atposition 25 is Asp or Asn; Xaa at position 26 is Thr or Asp; Xaa atposition 27 is Ser, Thr, Asn or Lys; Xaa at position 28 is Phe, Tyr orPro; Xaa at position 29 is Phe or Tyr; Xaa at position 30 is Ser, Gly orLys; Xaa at position 31 is Val, Ile or Met; Xaa at position 32 is Gly,Ala or Asp; Xaa at position 33 is Asn, Ser, Gln or Pro; Xaa at position35 is Lys, Glu or Ser; Xaa at position 36 is Gln, Asn or Ser; Xaa atposition 37 is Glu or Asp; Xaa at position 38 is Thr or Ser; Xaa atposition 42 is Ser or Asn; Xaa at position 44 is Ser, Asp, Ala or Leu;Xaa at position 47 is Phe or Tyr; Xaa at position 48 is Leu or Met; Xaaat position 49 is Leu or Met; Xaa at position 50 is Ser, Ala or Tyr; Xaaat position 51 is Leu or Val; Xaa at position 52 is Lys or Gln; Xaa atposition 53 is Lys, Arg, Met or Leu; Xaa at position 54 is Asn, Lys orGly; Xaa at position 55 is Gly or Ser; Xaa at position 56 is Ala, Thr,Gln or Ser; Xaa at position 57 is Gln, Val or Ala; Xaa at position 58 isHis, Ala, Lys, Tyr or Thr; Xaa at position 59 is Pro or Thr; Xaa atposition 62 is Val or Ile; Xaa at position 63 is Gln, Ser or Leu; Xaa atposition 64 is Ala, Gln or Ser; Xaa at position 65 is Ser or Thr; Xaa atposition 67 is Lys, Gln, Arg or Asn; Xaa at position 69 is Glu, Lys orVal; Xaa at position 70 is Val or Ile; Xaa at position 71 is Asp, Glu orTyr; Xaa at position 72 is Asn, His, Ser or Asp; Xaa at position 73 isAsn, Ser or Asp; Xaa at position 74 is Ala, Thr, Met, Ile or Lys; Xaa atposition 76 is Lys or Thr; Xaa at position 78 is Gln, His or Ser; Xaa atposition 80 is Arg, Glu or Gln; Xaa at position 81 is Leu, Pro, Ala orThr; Xaa at position 82 is Ile or Leu; Xaa at position 83 is Glu, His,Asn, Gln or Leu; Xaa at position 85 is Leu, Val or Ala; and Xaa atposition 86 is Ser, Ala, Tyr or Asn, and wherein 1 to 14 amino acids areoptionally deleted from the N-terminus and/or C-terminus of the PIP-72polypeptide and/or an amino acid is inserted between residue 24 and 25relative to SEQ ID NO: 847.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 847 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acidsubstitutions, in any combination, at residues designated by Xaa in SEQID NO: 847 compared to the native amino acid at the correspondingposition of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 847 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29amino acid substitutions, in any combination, at residues designated byXaa in SEQ ID NO: 847 compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 848, wherein Xaa at position 2 is Gly, Lys, Alaor Arg; Xaa at position 3 is Ile, Leu or Val; Xaa at position 4 is Thror Ser; Xaa at position 5 is Val, Ile or Leu; Xaa at position 6 is Thr,Lys, Ser or Arg; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thror Ala; Xaa at position 9 is Ser, Ala or Thr; Xaa at position 11 is Asn,Lys, Thr, Gln, Arg, His or Ser; Xaa at position 12 is Pro, Thr, Lys, Seror Arg; Xaa at position 13 is Ile, Val or Leu; Xaa at position 14 is Gluor Asp; Xaa at position 15 is Val, Ala, Ile or Leu; Xaa at position 16is Ala or Ser; Xaa at position 17 is Ile, Val or Leu; Xaa at position 18is Asn, Ser, Gln or Thr; Xaa at position 19 is His, Lys, Ala, Gln, Asnor Arg; Xaa at position 21 is Gly, Arg or Lys; Xaa at position 22 isSer, Lys, Asn, Thr, Arg, Asp, Glu or Gln; Xaa at position 25 is Asp,Asn, Glu or Gln; Xaa at position 26 is Thr, Asp, Ser or Glu; Xaa atposition 27 is Ser, Thr, Lys, Asn, Gln or Arg; Xaa at position 28 isPhe, Tyr, Pro or Trp; Xaa at position 29 is Phe, Tyr or Trp; Xaa atposition 30 is Ser, Gly, Lys, Thr or Arg; Xaa at position 31 is Val,Ile, Met or Leu; Xaa at position 32 is Gly, Ala, Asp or Glu; Xaa atposition 33 is Asn, Ser, Gln, Pro or Thr; Xaa at position 35 is Lys,Glu, Ser, Arg or Thr; Xaa at position 36 is Gln, Ser, Asn or Thr; Xaa atposition 37 is Glu or Asp; Xaa at position 38 is Thr or Ser; Xaa atposition 42 is Ser, Asn, Thr or Gln; Xaa at position 44 is Ser, Asp,Ala, Leu, Thr, Glu, Ile or Val; Xaa at position 47 is Phe, Tyr or Trp;Xaa at position 48 is Leu, Met, Ile or Val; Xaa at position 49 is Leu,Met, Ile or Val; Xaa at position 50 is Ser, Ala, Tyr or Thr; Xaa atposition 51 is Leu, Val or Ile; Xaa at position 52 is Lys, Gln, Arg orAsn; Xaa at position 53 is Lys, Arg, Met, Leu, Ile or Val; Xaa atposition 54 is Asn, Lys, Gly, Gln or Arg; Xaa at position 55 is Gly, Seror Thr; Xaa at position 56 is Ala, Thr, Gln, Ser or Asn; Xaa at position57 is Gln, Val, Ala, Asn, Leu or Ile; Xaa at position 58 is His, Ala,Lys, Tyr or Thr; Xaa at position 59 is Pro, Thr or Ser; Xaa at position62 is Val, Ile or Leu; Xaa at position 63 is Gln, Ser, Leu, Asn, Thr,Ile or Val; Xaa at position 64 is Ala, Gln, Ser, Asn or Thr; Xaa atposition 65 is Ser or Thr; Xaa at position 67 is Lys, Gln, Asn or Arg;Xaa at position 69 is Glu, Val, Asp, Lys, Arg, Ile or Leu; Xaa atposition 70 is Val, Ile or Leu; Xaa at position 71 is Asp, Glu, Tyr orTrp; Xaa at position 72 is Asn, His, Ser, Asp, Gln, Thr or Glu; Xaa atposition 73 is Asn, Ser, Asp, Gln, Thr or Glu; Xaa at position 74 isAla, Thr, Met, Ile, Lys, Ser, Leu, Val or Arg; Xaa at position 76 isLys, Thr, Arg or Ser; Xaa at position 78 is Gln, His, Ser, Asn or Thr;Xaa at position 80 is Arg, Glu, Gln, Lys, Asp or Asn; Xaa at position 81is Leu, Pro, Thr, Ile, Val, Ala or Ser; Xaa at position 82 is Ile, Leuor Val; Xaa at position 83 is Glu, His, Asn, Leu, Gln, Ile or Val; Xaaat position 85 is Leu, Val or Ala; and Xaa at position 86 is Ser, Ala,Tyr, Asn or Thr, and wherein 1 to 14 amino acids are optionally deletedfrom the N-terminus and/or C-terminus of the PIP-72 polypeptide and/oran amino acid is inserted between residue 24 and 25 relative to SEQ IDNO: 848.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 848 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acidsubstitutions, in any combination, at residues designated by Xaa in SEQID NO: 848 compared to the native amino acid at the correspondingposition of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 848 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29amino acid substitutions, in any combination, at residues designated byXaa in SEQ ID NO: 848 compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 849 wherein Xaa at position 2 is Gly, Ala, Cys,Asp, Glu, Ile, Lys, Leu, Asn, Arg, Ser, Thr, Val, Trp or Tyr; Xaa atposition 3 is Ile, Leu, Val or Trp; Xaa at position 4 is Thr, Ala, Asp,Glu, His, Ile, Lys, Leu, Arg, Ser, Val, Trp or Tyr; Xaa at position 5 isVal, Ala, Cys, Gly, His, Ile, Leu or Tyr; Xaa at position 6 is Thr, Ala,Cys, Phe, Gly, His, Ile, Lys, Met, Pro, Gln, Arg, Ser, Trp or Tyr; Xaaat position 7 is Asn, Ala or Val; Xaa at position 8 is Asn, Lys, Gly,Ser, Gln, Arg, Thr, Ala, Cys, Asp, Glu, His, Ile, Leu, Met or Val; Xaaat position 9 is Ser, Ala, Cys, Gly or Thr; Xaa at position 11 is Asn,Lys, Thr, Gln, Arg, Ser, Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, Met,Val or Tyr; Xaa at position 12 is Pro, Thr, Lys, Ser, Arg, Ala, Cys,Asp, Glu, Gly, His, Leu, Asn, Gln, Arg, Val, Trp or Tyr; Xaa at position13 is Ile, Asn, Gln, Leu or Val; Xaa at position 14 is Glu, Ala, Cys,Phe, His, Lys, Asp or Gln; Xaa at position 15 is Val, Ala, Ile, Leu,Cys, Met or Arg; Xaa at position 16 is Ala or Ser; Xaa at position 17 isIle, Glu, Leu or Val; Xaa at position 18 is Asn, Gln, Thr or Ser; Xaa atposition 19 is His, Lys, Ala, Arg, Glu, Leu, Pro, Ser or Tyr; Xaa atposition 20 is Trp, Ala or Thr; Xaa at position 21 is Gly, Arg or Lys;Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met,Asn, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position 23 is Asp, Ala,Gly, His, Lys, Met, Asn, Gln, Ser, Thr or Val; Xaa at position 24 isGly, Asp or Phe; Xaa at position 25 is Asp, Ala, Glu, Phe, Asn or Gln;Xaa at position 26 is Thr, Glu, Asp, Ser or Pro; Xaa at position 27 isSer, Thr, Lys, Arg, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn or Gln; Xaaat position 28 is Phe, Tyr, Pro or Trp; Xaa at position 29 is Phe, Ala,Cys, Ile, Leu, Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Gly,Lys, Thr, Arg, Ala, Cys, Asp, Glu, Phe, His, Leu, Met, Asn, Pro, Gln,Val, Trp or Tyr; Xaa at position 31 is Val, Ile, Met or Leu; Xaa atposition 32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro,Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 33 is Asn, Ser,Gln, Pro, Thr, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Arg,Val or Tyr; Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met,Asn, Gln, Arg, Ser, Thr or Tyr; Xaa at position 35 is Lys, Glu, Ala,Cys, Asp, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaaat position 36 is Gln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro,Arg, Ser, Thr or Val; Xaa at position 37 is Glu, Asp, Ala, Cys, Phe,Gly, Ile, Lys, Leu, Met, Asn, Ser, Thr or Val; Xaa at position 38 isThr, Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln,Arg, Val, Trp or Tyr; Xaa at position 39 is Trp or Phe; Xaa at position40 is Asp, Ala, Cys, Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 42 is Ser, Asn, Thr,Ala, Cys, Asp, Glu, Phe, Gly, Ile, Lys, Leu, Met, Arg, Val, Trp, Tyr orGln; Xaa at position 44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, Ala, Gly,Leu, Met, Asn, Pro, Gln, Val, Tyr or Val; Xaa at position 45 is Arg, Lysor Ser; Xaa at position 46 is Gly, Ala or Gln; Xaa at position 47 isPhe, Tyr Cys, Val or Trp; Xaa at position 48 is Leu, Met, Ile, Cys, Phe,Met, Arg, Tyr or Val; Xaa at position 49 is Leu, Met, Ile or Val; Xaa atposition 50 is Ser, Ala, Tyr, Cys, Asp, Ile, Met, Pro, Gln, Val or Thr;Xaa at position 51 is Leu, Val, Ala, Cys, Met or Ile; Xaa at position 52is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Gln, Trp orTyr; Xaa at position 53 is Lys, Arg, Met, Leu, Ile, Ala, Cys, Asp, Glu,Phe, His, Asn, Gln, Ser, Thr, Tyr or Val; Xaa at position 54 is Asn,Cys, Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position55 is Gly, Ser or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, Gly,Leu, Pro, Arg or Asn; Xaa at position 57 is Gln, Glu, Leu, Met, Ser,Val, Ala, Asn, Ile or Thr; Xaa at position 58 is His, Ala, Lys, Asp,Phe, Leu, Met, Asn, Arg, Trp, Tyr or Thr; Xaa at position 59 is Pro, Thror Ser; Xaa at position 60 is Tyr, Glu or Phe; Xaa at position 62 isVal, Ile or Leu; Xaa at position 63 is Gln, Ser, Cys, Gly, Ile, Leu,Met, Asn, Thr, Val or Tyr; Xaa at position 64 is Ala, Gln, Asn, Phe,Gly, His, Arg, Ser or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Leu, Asn, Val or Thr; Xaa at position 66 isSer, Ala or Gly; Xaa at position 67 is Lys, Gln, Asn or Arg; Xaa atposition 67 is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln,Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu orVal; Xaa at position 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met,Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Ile, Cys orLeu; Xaa at position 71 is Asp, Glu, Tyr, Ala, Cys, Gly, His, Ile, Leu,Met, Asn, Ser, Thr, Val or Trp; Xaa at position 72 is Asn, Ala, Cys,Asp, Glu, Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, His or Trp; Xaaat position 73 is Asn, Ser, Asp, Gln, Thr, Ala, Cys, Phe, Gly, His, Ile,Leu, Val, Tyr or Glu; Xaa at position 74 is Ala, Thr, Met, Ile, Lys,Ser, Leu, Val, Cys, Asp, Phe, Gly, His, Asn, Gln, Tyr or Arg; Xaa atposition 75 is Val, Cys, Ile or Leu; Xaa at position 76 is Lys, Ala,Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa atposition 77 is Asp Tyr; Xaa at position 78 is Gln, His, Ser, Asn, Ala,Cys, Asp, Phe, Gly, Ile, Leu, Met, Asn, Arg, Val, Tyr or Thr; Xaa atposition 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn,Gln, Arg, Ser, Thr, Trp or Tyr; Xaa at position 80 is Arg, Glu, Gln,Lys, Asp, Ala, Cys, Phe, Gly, His, Ile, Leu, Ser, Thr, Val, Tyr or Asn;Xaa at position 81 is Leu, Pro, Thr, Ile, Val, Ala, Cys, Asp, Phe, Gly,His or Ser; Xaa at position 82 is Ile, Ala, Leu, Met, Arg and Val; Xaaat position 83 is Glu, His, Asn, Leu, Gln, Ile, Ala, Cys, Asp, Phe, Gly,Lys, Pro, Arg, Ser, Thr, Tyr or Val; Xaa at position 84 is Pro, Ala,Cys, Glu, Ile, Ser, Val, Trp or Tyr; Xaa at position 85 is Leu, Val,Cys, Gly or Ala; and Xaa at position 86 is Ser, Ala, Tyr, Asn, Ile, Valor Thr, and wherein, 1 to 14 amino acids are optionally deleted from theN-terminus and/or C-terminus of the PIP-72 polypeptide and/or an aminoacid is inserted between residue 24 and 25 relative to SEQ ID NO: 849.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 849 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30,31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 or 45 amino acidsubstitutions, in any combination, at residues designated by Xaa in SEQID NO: 849 compared to the native amino acid at the correspondingposition of SEQ ID NO: 2.

In some embodiments a PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 849 having 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 11, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28 or 29amino acid substitutions, in any combination, at residues designated byXaa in SEQ ID NO: 849 compared to the native amino acid at thecorresponding position of SEQ ID NO: 2.

In some embodiments exemplary PIP-72 polypeptides are encoded by thepolynucleotide sequence set forth in SEQ ID NO: 1, SEQ ID NO: 3, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ IDNO: 17, SEQ ID NO: 27, SEQ ID NO: 31, any one of SEQ ID NO: 287-SEQ IDNO: 527, any one of SEQ ID NO: 796-SEQ ID NO: 815, SEQ ID NO: 769, SEQID NO: 770, SEQ ID NO: 850, SEQ ID NO: 852, any one of SEQ ID NO:853-SEQ ID NO: 864, any one of SEQ ID NO: 915-SEQ ID NO: 926, SEQ ID NO:949, SEQ ID NO: 950, SEQ ID NO: 954, SEQ ID NO: 955, SEQ ID NO: 956, SEQID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQ ID NO: 962, SEQ ID NO:963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO: 967 or SEQ ID NO: 968.

In some embodiments the PIP-72 polypeptide is encoded by thepolynucleotide of SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO:7, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO:27, SEQ ID NO: 31, SEQ ID NO: 769, SEQ ID NO: 770, SEQ ID NO: 850, SEQID NO: 852, SEQ ID NO: 949, SEQ ID NO: 950, SEQ ID NO: 954, SEQ ID NO:955, SEQ ID NO: 956, SEQ ID NO: 957, SEQ ID NO: 958, SEQ ID NO: 961, SEQID NO: 962, SEQ ID NO: 963, SEQ ID NO: 965, SEQ ID NO: 966, SEQ ID NO:967 or SEQ ID NO: 968.

In some embodiments exemplary PIP-72 polypeptides are set forth in SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14; SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32,SEQ ID NO: 528, SEQ ID NO: 529, SEQ ID NO: 530, SEQ ID NO: 531, SEQ IDNO: 532, SEQ ID NO: 533, SEQ ID NO: 534, SEQ ID NO: 535, SEQ ID NO: 536,SEQ ID NO: 537, SEQ ID NO: 538, SEQ ID NO: 539, SEQ ID NO: 540, SEQ IDNO: 541, SEQ ID NO: 542, SEQ ID NO: 543, SEQ ID NO: 544, SEQ ID NO: 545,SEQ ID NO: 546, SEQ ID NO: 547, SEQ ID NO: 548, SEQ ID NO: 549, SEQ IDNO: 550, SEQ ID NO: 551, SEQ ID NO: 552, SEQ ID NO: 553, SEQ ID NO: 554,SEQ ID NO: 555, SEQ ID NO: 556, SEQ ID NO: 557, SEQ ID NO: 558, SEQ IDNO: 559, SEQ ID NO: 560, SEQ ID NO: 561, SEQ ID NO: 562, SEQ ID NO: 563,SEQ ID NO: 564, SEQ ID NO: 565, SEQ ID NO: 566, SEQ ID NO: 567, SEQ IDNO: 568, SEQ ID NO: 569, SEQ ID NO: 570, SEQ ID NO: 571, SEQ ID NO: 572,SEQ ID NO: 573, SEQ ID NO: 574, SEQ ID NO: 575, SEQ ID NO: 576, SEQ IDNO: 577, SEQ ID NO: 578, SEQ ID NO: 579, SEQ ID NO: 580, SEQ ID NO: 581,SEQ ID NO: 582, SEQ ID NO: 583, SEQ ID NO: 584, SEQ ID NO: 585, SEQ IDNO: 586, SEQ ID NO: 587, SEQ ID NO: 588, SEQ ID NO: 589, SEQ ID NO: 590,SEQ ID NO: 591, SEQ ID NO: 592, SEQ ID NO: 593, SEQ ID NO: 594, SEQ IDNO: 595, SEQ ID NO: 596, SEQ ID NO: 597, SEQ ID NO: 598, SEQ ID NO: 599,SEQ ID NO: 600, SEQ ID NO: 601, SEQ ID NO: 602, SEQ ID NO: 603, SEQ IDNO: 604, SEQ ID NO: 605, SEQ ID NO: 606, SEQ ID NO: 607, SEQ ID NO: 608,SEQ ID NO: 609, SEQ ID NO: 610, SEQ ID NO: 611, SEQ ID NO: 612, SEQ IDNO: 613, SEQ ID NO: 614, SEQ ID NO: 615, SEQ ID NO: 616, SEQ ID NO: 617,SEQ ID NO: 618, SEQ ID NO: 619, SEQ ID NO: 620, SEQ ID NO: 621, SEQ IDNO: 622, SEQ ID NO: 623, SEQ ID NO: 624, SEQ ID NO: 625, SEQ ID NO: 626,SEQ ID NO: 627, SEQ ID NO: 628, SEQ ID NO: 629, SEQ ID NO: 630, SEQ IDNO: 631, SEQ ID NO: 632, SEQ ID NO: 633, SEQ ID NO: 634, SEQ ID NO: 635,SEQ ID NO: 636, SEQ ID NO: 637, SEQ ID NO: 638, SEQ ID NO: 639, SEQ IDNO: 640, SEQ ID NO: 641, SEQ ID NO: 642, SEQ ID NO: 643, SEQ ID NO: 644,SEQ ID NO: 645, SEQ ID NO: 646, SEQ ID NO: 647, SEQ ID NO: 648, SEQ IDNO: 649, SEQ ID NO: 650, SEQ ID NO: 651, SEQ ID NO: 652, SEQ ID NO: 653,SEQ ID NO: 654, SEQ ID NO: 655, SEQ ID NO: 656, SEQ ID NO: 657, SEQ IDNO: 658, SEQ ID NO: 659, SEQ ID NO: 660, SEQ ID NO: 661, SEQ ID NO: 662,SEQ ID NO: 663, SEQ ID NO: 664, SEQ ID NO: 665, SEQ ID NO: 666, SEQ IDNO: 667, SEQ ID NO: 668, SEQ ID NO: 669, SEQ ID NO: 670, SEQ ID NO: 671,SEQ ID NO: 672, SEQ ID NO: 673, SEQ ID NO: 674, SEQ ID NO: 675, SEQ IDNO: 676, SEQ ID NO: 677, SEQ ID NO: 678, SEQ ID NO: 679, SEQ ID NO: 680,SEQ ID NO: 681, SEQ ID NO: 682, SEQ ID NO: 683, SEQ ID NO: 684, SEQ IDNO: 685, SEQ ID NO: 686, SEQ ID NO: 687, SEQ ID NO: 688, SEQ ID NO: 689,SEQ ID NO: 690, SEQ ID NO: 691, SEQ ID NO: 692, SEQ ID NO: 693, SEQ IDNO: 694, SEQ ID NO: 695, SEQ ID NO: 696, SEQ ID NO: 697, SEQ ID NO: 698,SEQ ID NO: 699, SEQ ID NO: 700, SEQ ID NO: 701, SEQ ID NO: 702, SEQ IDNO: 703, SEQ ID NO: 704, SEQ ID NO: 705, SEQ ID NO: 706, SEQ ID NO: 707,SEQ ID NO: 708, SEQ ID NO: 709, SEQ ID NO: 710, SEQ ID NO: 711, SEQ IDNO: 712, SEQ ID NO: 713, SEQ ID NO: 714, SEQ ID NO: 715, SEQ ID NO: 716,SEQ ID NO: 717, SEQ ID NO: 718, SEQ ID NO: 719, SEQ ID NO: 720, SEQ IDNO: 721, SEQ ID NO: 722, SEQ ID NO: 723, SEQ ID NO: 724, SEQ ID NO: 725,SEQ ID NO: 726, SEQ ID NO: 727, SEQ ID NO: 728, SEQ ID NO: 729, SEQ IDNO: 730, SEQ ID NO: 731, SEQ ID NO: 732, SEQ ID NO: 733, SEQ ID NO: 734,SEQ ID NO: 735, SEQ ID NO: 736, SEQ ID NO: 737, SEQ ID NO: 738, SEQ IDNO: 739, SEQ ID NO: 740, SEQ ID NO: 741, SEQ ID NO: 742, SEQ ID NO: 743,SEQ ID NO: 744, SEQ ID NO: 745, SEQ ID NO: 746, SEQ ID NO: 747, SEQ IDNO: 748, SEQ ID NO: 749, SEQ ID NO: 750, SEQ ID NO: 751, SEQ ID NO: 752,SEQ ID NO: 753, SEQ ID NO: 754, SEQ ID NO: 755, SEQ ID NO: 756, SEQ IDNO: 757, SEQ ID NO: 758, SEQ ID NO: 759, SEQ ID NO: 760, SEQ ID NO: 761,SEQ ID NO: 762, SEQ ID NO: 763, SEQ ID NO: 764, SEQ ID NO: 765, SEQ IDNO: 766, SEQ ID NO: 767, SEQ ID NO: 768, SEQ ID NO: 771, SEQ ID NO: 772,SEQ ID NO: 825, SEQ ID NO: 826, SEQ ID NO: 827, SEQ ID NO: 828, SEQ IDNO: 829, SEQ ID NO: 830, SEQ ID NO: 831, SEQ ID NO: 832, SEQ ID NO: 833,SEQ ID NO: 834, SEQ ID NO: 835, SEQ ID NO: 836, SEQ ID NO: 837, SEQ IDNO: 838, SEQ ID NO: 839, SEQ ID NO: 840, SEQ ID NO: 841, SEQ ID NO: 842,SEQ ID NO: 843, SEQ ID NO: 844, SEQ ID NO: 852, SEQ ID NO: 853, SEQ IDNO: 854, SEQ ID NO: 855, SEQ ID NO: 856, SEQ ID NO: 857, SEQ ID NO: 858,SEQ ID NO: 859, SEQ ID NO: 860, SEQ ID NO: 861, SEQ ID NO: 862, SEQ IDNO: 863, SEQ ID NO: 864, SEQ ID NO: 903, SEQ ID NO: 904, SEQ ID NO: 905,SEQ ID NO: 906, SEQ ID NO: 907, SEQ ID NO: 908, SEQ ID NO: 909, SEQ IDNO: 910, SEQ ID NO: 911, SEQ ID NO: 912, SEQ ID NO: 913, SEQ ID NO: 914,SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ IDNO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940,SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945 and SEQID NO: 946.

In some embodiments the PIP-72 polypeptide comprises an amino acidsequence of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14; SEQ ID NO: 18, SEQ ID NO: 28,SEQ ID NO: 32, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ IDNO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939,SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ IDNO: 945 or SEQ ID NO: 946.

In some embodiments exemplary PIP-72 polypeptides are the polypeptidesshown in Table 14, Table 17, Table 20, Table 23, Table 24, Table 26,Table 28, and/or Table 29 and any combinations of the amino acidsubstitutions thereof as well as deletions and or insertions andfragments thereof.

In some embodiments a PIP-72 polypeptide has a calculated molecularweight of between about 6 kDa and about 13 kDa between about 7 kDa andabout 12 kDa, between about 8 kDa and about 11 kDa, between about 9 kDaand about 10 kDa, about 8.75 kDa, about 9 kDa, about 9.25 kDa, about 9.5kDa, about 9.75 kDa, about 10 kDa, about 10.25 kDa, and about 10.5 kDa.As used herein, the term “about” used in the context of molecular weightof a PIP-72 polypeptide means±0.25 kilodaltons.

In some embodiments the PIP-72 polypeptide has a modified physicalproperty. As used herein, the term “physical property” refers to anyparameter suitable for describing the physical-chemical characteristicsof a protein. As used herein, “physical property of interest” and“property of interest” are used interchangeably to refer to physicalproperties of proteins that are being investigated and/or modified.Examples of physical properties include, but are not limited to netsurface charge and charge distribution on the protein surface, nethydrophobicity and hydrophobic residue distribution on the proteinsurface, surface charge density, surface hydrophobicity density, totalcount of surface ionizable groups, surface tension, protein size and itsdistribution in solution, melting temperature, heat capacity, and secondvirial coefficient. Examples of physical properties also include, butare not limited to solubility, folding, stability, and digestibility. Insome embodiments the PIP-72 polypeptide has increased digestibility ofproteolytic fragments in an insect gut. Models for digestion bysimulated simulated gastric fluids are known to one skilled in the art(Fuchs, R. L. and J. D. Astwood. Food Technology 50: 83-88, 1996;Astwood, J. D., et al Nature Biotechnology 14: 1269-1273, 1996; Fu T Jet al J. Agric Food Chem. 50: 7154-7160, 2002).

In some embodiments variants include polypeptides that differ in aminoacid sequence due to mutagenesis. Variant proteins encompassed by thedisclosure are biologically active, that is they continue to possess thedesired biological activity (i.e. pesticidal activity) of the nativeprotein. In some embodiment the variant will have at least about 10%, atleast about 30%, at least about 50%, at least about 70%, at least about80% or more of the insecticidal activity of the native protein. In someembodiments, the variants may have improved activity over the nativeprotein.

Bacterial genes quite often possess multiple methionine initiationcodons in proximity to the start of the open reading frame. Often,translation initiation at one or more of these start codons will lead togeneration of a functional protein. These start codons can include ATGcodons. However, bacteria such as Bacillus sp. also recognize the codonGTG as a start codon, and proteins that initiate translation at GTGcodons contain a methionine at the first amino acid. On rare occasions,translation in bacterial systems can initiate at a TTG codon, though inthis event the TTG encodes a methionine. Furthermore, it is not oftendetermined a priori which of these codons are used naturally in thebacterium. Thus, it is understood that use of one of the alternatemethionine codons may also lead to generation of pesticidal proteins.These pesticidal proteins are encompassed in the present disclosure andmay be used in the methods of the present disclosure. It will beunderstood that, when expressed in plants, it will be necessary to alterthe alternate start codon to ATG for proper translation.

In another aspect the PIP-72 polypeptide may be expressed as a precursorprotein with an intervening sequence that catalyzes multi-step, posttranslational protein splicing. Protein splicing involves the excisionof an intervening sequence from a polypeptide with the concomitantjoining of the flanking sequences to yield a new polypeptide (Chong, etal., (1996) J. Biol. Chem., 271:22159-22168). This intervening sequenceor protein splicing element, referred to as inteins, which catalyzetheir own excision through three coordinated reactions at the N-terminaland C-terminal splice junctions: an acyl rearrangement of the N-terminalcysteine or serine; a transesterfication reaction between the twotermini to form a branched ester or thioester intermediate and peptidebond cleavage coupled to cyclization of the intein C-terminal asparagineto free the intein (Evans, et al., (2000) J. Biol. Chem., 275:9091-9094.The elucidation of the mechanism of protein splicing has led to a numberof intein-based applications (Comb, et al., U.S. Pat. No. 5,496,714;Comb, et al., U.S. Pat. No. 5,834,247; Camarero and Muir, (1999) J.Amer. Chem. Soc. 121:5597-5598; Chong, et al., (1997) Gene 192:271-281,Chong, et al., (1998) Nucleic Acids Res. 26:5109-5115; Chong, et al.,(1998) J. Biol. Chem. 273:10567-10577; Cotton, et al., (1999) J. Am.Chem. Soc. 121:1100-1101; Evans, et al., (1999) J. Biol. Chem.274:18359-18363; Evans, et al., (1999) J. Biol. Chem. 274:3923-3926;Evans, et al., (1998) Protein Sci. 7:2256-2264; Evans, et al., (2000) J.Biol. Chem. 275:9091-9094; Iwai and Pluckthun, (1999) FEBS Lett.459:166-172; Mathys, et al., (1999) Gene 231:1-13; Mills, et al., (1998)Proc. Natl. Acad. Sci. USA 95:3543-3548; Muir, et al., (1998) Proc.Natl. Acad. Sci. USA 95:6705-6710; Otomo, et al., (1999) Biochemistry38:16040-16044; Otomo, et al., (1999) J. Biolmol. NMR 14:105-114; Scott,et al., (1999) Proc. Natl. Acad. Sci. USA 96:13638-13643; Severinov andMuir, (1998) J. Biol. Chem. 273:16205-16209; Shingledecker, et al.,(1998) Gene 207:187-195; Southworth, et al., (1998) EMBO J. 17:918-926;Southworth, et al., (1999) Biotechniques 27:110-120; Wood, et al.,(1999) Nat. Biotechnol. 17:889-892; Wu, et al., (1998a) Proc. Natl.Acad. Sci. USA 95:9226-9231; Wu, et al., (1998b) Biochim Biophys Acta1387:422-432; Xu, et al., (1999) Proc. Natl. Acad. Sci. USA 96:388-393;Yamazaki, et al., (1998) J. Am. Chem. Soc., 120:5591-5592). For theapplication of inteins in plant transgenes, see, Yang, et al.,(Transgene Res 15:583-593 (2006)) and Evans, et al., (Annu. Rev. PlantBiol. 56:375-392 (2005)).

In another aspect the PIP-72 polypeptide may be encoded by two separategenes where the intein of the precursor protein comes from the twogenes, referred to as a split-intein, and the two portions of theprecursor are joined by a peptide bond formation. This peptide bondformation is accomplished by intein-mediated trans-splicing. For thispurpose, a first and a second expression cassette comprising the twoseparate genes further code for inteins capable of mediating proteintrans-splicing. By trans-splicing, the proteins and polypeptides encodedby the first and second fragments may be linked by peptide bondformation. Trans-splicing inteins may be selected from the nucleolar andorganellar genomes of different organisms including eukaryotes,archaebacteria and eubacteria. Inteins that may be used for are listedat neb.com/neb/inteins.html, which can be accessed on the world-wide webusing the “www” prefix). The nucleotide sequence coding for an inteinmay be split into a 5′ and a 3′ part that code for the 5′ and the 3′part of the intein, respectively. Sequence portions not necessary forintein splicing (e.g. homing endonuclease domain) may be deleted. Theintein coding sequence is split such that the 5′ and the 3′ parts arecapable of trans-splicing. For selecting a suitable splitting site ofthe intein coding sequence, the considerations published by Southworth,et al., (1998) EMBO J. 17:918-926 may be followed. In constructing thefirst and the second expression cassette, the 5′ intein coding sequenceis linked to the 3′ end of the first fragment coding for the N-terminalpart of the PIP-72 polypeptide and the 3′ intein coding sequence islinked to the 5′ end of the second fragment coding for the C-terminalpart of the PIP-72 polypeptide.

In general, the trans-splicing partners can be designed using any splitintein, including any naturally-occurring or artificially-split splitintein. Several naturally-occurring split inteins are known, forexample: the split intein of the DnaE gene of Synechocystis sp. PCC6803(see, Wu, et al., (1998) Proc Natl Acad Sci USA. 95(16):9226-31 andEvans, et al., (2000) J Biol Chem. 275(13):9091-4 and of the DnaE genefrom Nostoc punctiforme (see, Iwai, et al., (2006) FEBS Lett.580(7):1853-8). Non-split inteins have been artificially split in thelaboratory to create new split inteins, for example: the artificiallysplit Ssp DnaB intein (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32) and split Sce VMA intein (see, Brenzel, et al., (2006)Biochemistry. 45(6):1571-8) and an artificially split fungal mini-intein(see, Elleuche, et al., (2007) Biochem Biophys Res Commun.355(3):830-4). There are also intein databases available that catalogueknown inteins (see for example the online-database available at:bioinformatics.weizmann.ac.il/{tilde over( )}pietro/inteins/Inteinstable.html, which can be accessed on theworld-wide web using the “www” prefix).

Naturally-occurring non-split inteins may have endonuclease or otherenzymatic activities that can typically be removed when designing anartificially-split split intein. Such mini-inteins or minimized splitinteins are well known in the art and are typically less than 200 aminoacid residues long (see, Wu, et al., (1998) Biochim Biophys Acta.1387:422-32). Suitable split inteins may have other purificationenabling polypeptide elements added to their structure, provided thatsuch elements do not inhibit the splicing of the split intein or areadded in a manner that allows them to be removed prior to splicing.Protein splicing has been reported using proteins that comprisebacterial intein-like (BIL) domains (see, Amitai, et al., (2003) MolMicrobiol. 47:61-73) and hedgehog (Hog) auto-processing domains (thelatter is combined with inteins when referred to as the Hog/inteinsuperfamily or HINT family (see, Dassa, et al., (2004) J Biol Chem.279:32001-7) and domains such as these may also be used to prepareartificially-split inteins. In particular, non-splicing members of suchfamilies may be modified by molecular biology methodologies to introduceor restore splicing activity in such related species. Recent studiesdemonstrate that splicing can be observed when a N-terminal split inteincomponent is allowed to react with a C-terminal split intein componentnot found in nature to be its “partner”; for example, splicing has beenobserved utilizing partners that have as little as 30 to 50% homologywith the “natural” splicing partner (see, Dassa, et al., (2007)Biochemistry. 46(1):322-30). Other such mixtures of disparate splitintein partners have been shown to be unreactive one with another (see,Brenzel, et al., (2006) Biochemistry. 45(6):1571-8). However, it iswithin the ability of a person skilled in the relevant art to determinewhether a particular pair of polypeptides is able to associate with eachother to provide a functional intein, using routine methods and withoutthe exercise of inventive skill.

In another aspect the PIP-72 polypeptide is a circular permuted variant.In certain embodiments the PIP-72 polypeptide is a circular permutedvariant of the polypeptide of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6,SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO:18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO:768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ IDNO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903-SEQ ID NO: 914, SEQID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO:934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ IDNO: 946.

The development of recombinant DNA methods has made it possible to studythe effects of sequence transposition on protein folding, structure andfunction. The approach used in creating new sequences resembles that ofnaturally occurring pairs of proteins that are related by linearreorganization of their amino acid sequences (Cunningham, et al., (1979)Proc. Natl. Acad. Sci. U.S.A. 76:3218-3222; Teather and Erfle, (1990) J.Bacteriol. 172:3837-3841; Schimming, et al., (1992) Eur. J. Biochem.204:13-19; Yamiuchi and Minamikawa, (1991) FEBS Lett. 260:127-130;MacGregor, et al., (1996) FEBS Lett. 378:263-266). The first in vitroapplication of this type of rearrangement to proteins was described byGoldenberg and Creighton (J. Mol. Biol. 165:407-413, 1983). In creatinga circular permuted variant a new N-terminus is selected at an internalsite (breakpoint) of the original sequence, the new sequence having thesame order of amino acids as the original from the breakpoint until itreaches an amino acid that is at or near the original C-terminus. Atthis point the new sequence is joined, either directly or through anadditional portion of sequence (linker), to an amino acid that is at ornear the original N-terminus and the new sequence continues with thesame sequence as the original until it reaches a point that is at ornear the amino acid that was N-terminal to the breakpoint site of theoriginal sequence, this residue forming the new C-terminus of the chain.The length of the amino acid sequence of the linker can be selectedempirically or with guidance from structural information or by using acombination of the two approaches. When no structural information isavailable, a small series of linkers can be prepared for testing using adesign whose length is varied in order to span a range from 0 to 50 Åand whose sequence is chosen in order to be consistent with surfaceexposure (hydrophilicity, Hopp and Woods, (1983) Mol. Immunol.20:483-489; Kyte and Doolittle, (1982) J. Mol. Biol. 157:105-132;solvent exposed surface area, Lee and Richards, (1971) J. Mol. Biol.55:379-400) and the ability to adopt the necessary conformation withoutderanging the configuration of the pesticidal polypeptide(conformationally flexible; Karplus and Schulz, (1985)Naturwissenschaften 72:212-213). Assuming an average of translation of2.0 to 3.8 Å per residue, this would mean the length to test would bebetween 0 to 30 residues, with 0 to 15 residues being the preferredrange. Exemplary of such an empirical series would be to constructlinkers using a cassette sequence such as Gly-Gly-Gly-Ser repeated ntimes, where n is 1, 2, 3 or 4. Those skilled in the art will recognizethat there are many such sequences that vary in length or compositionthat can serve as linkers with the primary consideration being that theybe neither excessively long nor short (cf., Sandhu, (1992) Critical Rev.Biotech. 12:437-462); if they are too long, entropy effects will likelydestabilize the three-dimensional fold, and may also make foldingkinetically impractical, and if they are too short, they will likelydestabilize the molecule because of torsional or steric strain. Thoseskilled in the analysis of protein structural information will recognizethat using the distance between the chain ends, defined as the distancebetween the c-alpha carbons, can be used to define the length of thesequence to be used or at least to limit the number of possibilitiesthat must be tested in an empirical selection of linkers. They will alsorecognize that it is sometimes the case that the positions of the endsof the polypeptide chain are ill-defined in structural models derivedfrom x-ray diffraction or nuclear magnetic resonance spectroscopy data,and that when true, this situation will therefore need to be taken intoaccount in order to properly estimate the length of the linker required.From those residues whose positions are well defined are selected tworesidues that are close in sequence to the chain ends, and the distancebetween their c-alpha carbons is used to calculate an approximate lengthfor a linker between them. Using the calculated length as a guide,linkers with a range of number of residues (calculated using 2 to 3.8 Åper residue) are then selected. These linkers may be composed of theoriginal sequence, shortened or lengthened as necessary, and whenlengthened the additional residues may be chosen to be flexible andhydrophilic as described above; or optionally the original sequence maybe substituted for using a series of linkers, one example being theGly-Gly-Gly-Ser cassette approach mentioned above; or optionally acombination of the original sequence and new sequence having theappropriate total length may be used. Sequences of pesticidalpolypeptides capable of folding to biologically active states can beprepared by appropriate selection of the beginning (amino terminus) andending (carboxyl terminus) positions from within the originalpolypeptide chain while using the linker sequence as described above.Amino and carboxyl termini are selected from within a common stretch ofsequence, referred to as a breakpoint region, using the guidelinesdescribed below. A novel amino acid sequence is thus generated byselecting amino and carboxyl termini from within the same breakpointregion. In many cases the selection of the new termini will be such thatthe original position of the carboxyl terminus immediately preceded thatof the amino terminus. However, those skilled in the art will recognizethat selections of termini anywhere within the region may function, andthat these will effectively lead to either deletions or additions to theamino or carboxyl portions of the new sequence. It is a central tenet ofmolecular biology that the primary amino acid sequence of a proteindictates folding to the three-dimensional structure necessary forexpression of its biological function. Methods are known to thoseskilled in the art to obtain and interpret three-dimensional structuralinformation using x-ray diffraction of single protein Crystals ornuclear magnetic resonance spectroscopy of protein solutions. Examplesof structural information that are relevant to the identification ofbreakpoint regions include the location and type of protein secondarystructure (alpha and 3-10 helices, parallel and anti-parallel betasheets, chain reversals and turns, and loops; Kabsch and Sander, (1983)Biopolymers 22:2577-2637; the degree of solvent exposure of amino acidresidues, the extent and type of interactions of residues with oneanother (Chothia, (1984) Ann. Rev. Biochem. 53:537-572) and the staticand dynamic distribution of conformations along the polypeptide chain(Alber and Mathews, (1987) Methods Enzymol. 154:511-533). In some casesadditional information is known about solvent exposure of residues; oneexample is a site of post-translational attachment of carbohydrate whichis necessarily on the surface of the protein. When experimentalstructural information is not available or is not feasible to obtain,methods are also available to analyze the primary amino acid sequence inorder to make predictions of protein tertiary and secondary structure,solvent accessibility and the occurrence of turns and loops. Biochemicalmethods are also sometimes applicable for empirically determiningsurface exposure when direct structural methods are not feasible; forexample, using the identification of sites of chain scission followinglimited proteolysis in order to infer surface exposure (Gentile andSalvatore, (1993) Eur. J. Biochem. 218:603-621). Thus using either theexperimentally derived structural information or predictive methods(e.g., Srinivisan and Rose, (1995) Proteins: Struct., Funct. & Genetics22:81-99) the parental amino acid sequence is inspected to classifyregions according to whether or not they are integral to the maintenanceof secondary and tertiary structure. The occurrence of sequences withinregions that are known to be involved in periodic secondary structure(alpha and 3-10 helices, parallel and anti-parallel beta sheets) areregions that should be avoided. Similarly, regions of amino acidsequence that are observed or predicted to have a low degree of solventexposure are more likely to be part of the so-called hydrophobic core ofthe protein and should also be avoided for selection of amino andcarboxyl termini. In contrast, those regions that are known or predictedto be in surface turns or loops, and especially those regions that areknown not to be required for biological activity, are the preferredsites for location of the extremes of the polypeptide chain. Continuousstretches of amino acid sequence that are preferred based on the abovecriteria are referred to as a breakpoint region. Polynucleotidesencoding circular permuted PIP-72 polypeptides with newN-terminus/C-terminus which contain a linker region separating theoriginal C-terminus and N-terminus can be made essentially following themethod described in Mullins, et al., (1994) J. Am. Chem. Soc.116:5529-5533. Multiple steps of polymerase chain reaction (PCR)amplifications are used to rearrange the DNA sequence encoding theprimary amino acid sequence of the protein. Polynucleotides encodingcircular permuted PIP-72 polypeptides with new N-terminus/C-terminuswhich contain a linker region separating the original C-terminus andN-terminus can be made based on the tandem-duplication method describedin Horlick, et al., (1992) Protein Eng. 5:427-431. Polymerase chainreaction (PCR) amplification of the new N-terminus/C-terminus genes isperformed using a tandemly duplicated template DNA.

In another aspect fusion proteins are provided that include within itsamino acid sequence an amino acid sequence comprising a PIP-72polypeptide including but not limited to the polypeptide of SEQ ID NO:2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO:12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any oneof SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO:844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 846, SEQ ID NO: 847, SEQID NO: 848, SEQ ID NO: 849, SEQ ID NO: 852, any one of SEQ ID NO:903-SEQ ID NO: 914, any one of SEQ ID NO: 927-SEQ ID NO: 948, and activefragments thereof.

Methods for design and construction of fusion proteins (andpolynucleotides encoding same) are known to those of skill in the art.Polynucleotides encoding a PIP-72 polypeptide may be fused to signalsequences which will direct the localization of the PIP-72 polypeptideto particular compartments of a prokaryotic or eukaryotic cell and/ordirect the secretion of the PIP-72 polypeptide of the embodiments from aprokaryotic or eukaryotic cell. For example, in E. coli, one may wish todirect the expression of the protein to the periplasmic space. Examplesof signal sequences or proteins (or fragments thereof) to which thePIP-72 polypeptide may be fused in order to direct the expression of thepolypeptide to the periplasmic space of bacteria include, but are notlimited to, the pelB signal sequence, the maltose binding protein (MBP)signal sequence, MBP, the ompA signal sequence, the signal sequence ofthe periplasmic E. coli heat-labile enterotoxin B-subunit and the signalsequence of alkaline phosphatase. Several vectors are commerciallyavailable for the construction of fusion proteins which will direct thelocalization of a protein, such as the pMAL series of vectors(particularly the pMAL-p series) available from New England Biolabs®(240 County Road, Ipswich, Mass. 01938-2723). In a specific embodiment,the PIP-72 polypeptide may be fused to the pelB pectate lyase signalsequence to increase the efficiency of expression and purification ofsuch polypeptides in Gram-negative bacteria (see, U.S. Pat. Nos.5,576,195 and 5,846,818). Plant plastid transit peptide/polypeptidefusions are well known in the art (see, U.S. Pat. No. 7,193,133).Apoplast transit peptides such as rice or barley alpha-amylase secretionsignal are also well known in the art. The plastid transit peptide isgenerally fused N-terminal to the polypeptide to be targeted (e.g., thefusion partner). In one embodiment, the fusion protein consistsessentially of the plastid transit peptide and the PIP-72 polypeptide tobe targeted. In another embodiment, the fusion protein comprises theplastid transit peptide and the polypeptide to be targeted. In suchembodiments, the plastid transit peptide is preferably at the N-terminusof the fusion protein. However, additional amino acid residues may beN-terminal to the plastid transit peptide providing that the fusionprotein is at least partially targeted to a plastid. In a specificembodiment, the plastid transit peptide is in the N-terminal half,N-terminal third or N-terminal quarter of the fusion protein. Most orall of the plastid transit peptide is generally cleaved from the fusionprotein upon insertion into the plastid. The position of cleavage mayvary slightly between plant species, at different plant developmentalstages, as a result of specific intercellular conditions or theparticular combination of transit peptide/fusion partner used. In oneembodiment, the plastid transit peptide cleavage is homogenous such thatthe cleavage site is identical in a population of fusion proteins. Inanother embodiment, the plastid transit peptide is not homogenous, suchthat the cleavage site varies by 1-10 amino acids in a population offusion proteins. The plastid transit peptide can be recombinantly fusedto a second protein in one of several ways. For example, a restrictionendonuclease recognition site can be introduced into the nucleotidesequence of the transit peptide at a position corresponding to itsC-terminal end and the same or a compatible site can be engineered intothe nucleotide sequence of the protein to be targeted at its N-terminalend. Care must be taken in designing these sites to ensure that thecoding sequences of the transit peptide and the second protein are kept“in frame” to allow the synthesis of the desired fusion protein. In somecases, it may be preferable to remove the initiator methionine codon ofthe second protein when the new restriction site is introduced. Theintroduction of restriction endonuclease recognition sites on bothparent molecules and their subsequent joining through recombinant DNAtechniques may result in the addition of one or more extra amino acidsbetween the transit peptide and the second protein. This generally doesnot affect targeting activity as long as the transit peptide cleavagesite remains accessible and the function of the second protein is notaltered by the addition of these extra amino acids at its N-terminus.Alternatively, one skilled in the art can create a precise cleavage sitebetween the transit peptide and the second protein (with or without itsinitiator methionine) using gene synthesis (Stemmer, et al., (1995) Gene164:49-53) or similar methods. In addition, the transit peptide fusioncan intentionally include amino acids downstream of the cleavage site.The amino acids at the N-terminus of the mature protein can affect theability of the transit peptide to target proteins to plastids and/or theefficiency of cleavage following protein import. This may be dependenton the protein to be targeted. See, e.g., Comai, et al., (1988) J. Biol.Chem. 263(29):15104-9.

In some embodiments fusion proteins are provide comprising a PIP-72polypeptide, and an insecticdal polypeptide joined by an amino acidlinker.

In some embodiments fusion proteins are provided represented by aformula selected from the group consisting of:

R¹-L-R², R²-L-R¹, R¹-R² or R²-R¹

wherein R¹ is a PIP-72 polypeptide or the polypeptide of SEQ ID NO: 2,SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12,SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one ofSEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844,SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO:903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO:939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQID NO: 945, or SEQ ID NO: 946, R² is an insecticidal polypeptide. The R¹polypeptide is fused either directly or through a linker (L) segment tothe R² polypeptide. The term “directly” defines fusions in which thepolypeptides are joined without a peptide linker. Thus “L” represents achemical bound or polypeptide segment to which both R¹ and R² are fusedin frame, most commonly L is a linear peptide to which R¹ and R² arebound by amide bonds linking the carboxy terminus of R¹ to the aminoterminus of L and carboxy terminus of L to the amino terminus of R². By“fused in frame” is meant that there is no translation termination ordisruption between the reading frames of R¹ and R². The linking group(L) is generally a polypeptide of between 1 and 500 amino acids inlength. The linkers joining the two molecules are preferably designed to(1) allow the two molecules to fold and act independently of each other,(2) not have a propensity for developing an ordered secondary structurewhich could interfere with the functional domains of the two proteins,(3) have minimal hydrophobic or charged characteristic which couldinteract with the functional protein domains and (4) provide stericseparation of R¹ and R² such that R¹ and R² could interactsimultaneously with their corresponding receptors on a single cell.Typically surface amino acids in flexible protein regions include Gly,Asn and Ser. Virtually any permutation of amino acid sequencescontaining Gly, Asn and Ser would be expected to satisfy the abovecriteria for a linker sequence. Other neutral amino acids, such as Thrand Ala, may also be used in the linker sequence. Additional amino acidsmay also be included in the linkers due to the addition of uniquerestriction sites in the linker sequence to facilitate construction ofthe fusions.

In some embodiments the linkers comprise sequences selected from thegroup of formulas: (Gly₃Ser)_(n), (Gly₄Ser)_(n), (Gly₅Ser)_(n),(Gly_(n)Ser)_(n) or (AlaGlySer)_(n) where n is an integer. One exampleof a highly-flexible linker is the (GlySer)-rich spacer region presentwithin the pill protein of the filamentous bacteriophages, e.g.bacteriophages M13 or fd (Schaller, et al., 1975). This region providesa long, flexible spacer region between two domains of the pill surfaceprotein. Also included are linkers in which an endopeptidase recognitionsequence is included. Such a cleavage site may be valuable to separatethe individual components of the fusion to determine if they areproperly folded and active in vitro. Examples of various endopeptidasesinclude, but are not limited to, Plasmin, Enterokinase, Kallikerin,Urokinase, Tissue Plasminogen activator, clostripain, Chymosin,Collagenase, Russell's Viper Venom Protease, Postproline cleavageenzyme, V8 protease, Thrombin and factor Xa. In some embodiments thelinker comprises the amino acids EEKKN (SEQ ID NO: 488) from themulti-gene expression vehicle (MGEV), which is cleaved by vacuolarproteases as disclosed in US Patent Application Publication Number US2007/0277263. In other embodiments, peptide linker segments from thehinge region of heavy chain immunoglobulins IgG, IgA, IgM, IgD or IgEprovide an angular relationship between the attached polypeptides.Especially useful are those hinge regions where the cysteines arereplaced with serines. Linkers of the present disclosure includesequences derived from murine IgG gamma 2b hinge region in which thecysteines have been changed to serines. The fusion proteins are notlimited by the form, size or number of linker sequences employed and theonly requirement of the linker is that functionally it does notinterfere adversely with the folding and function of the individualmolecules of the fusion.

In another aspect chimeric PIP-72 polypeptides are provided that arecreated through joining two or more portions of PIP-72 genes, whichoriginally encoded separate PIP-72 proteins to create a chimeric gene.The translation of the chimeric gene results in a single chimeric PIP-72polypeptide with regions, motifs or domains derived from each of theoriginal polypeptides. In certain embodiments the chimeric proteincomprises portions, motifs or domains of PIP-72Aa (SEQ ID NO: 2),PIP-72Ba (SEQ ID NO: 4), PIP-72Ca (SEQ ID NO: 6) and PIP-72Cb (SEQ IDNO: 8), PIP-72 Da (SEQ ID NO: 10), PIP-72Db (SEQ ID NO: 12), PIP-72Dc(SEQ ID NO: 14), PIP-72Fa (SEQ ID NO: 18), PIP-72Ff (SEQ ID NO: 28) andPIP-72Gb (SEQ ID NO: 32), PIP-72Ab (SEQ ID NO: 927), PIP-72Bb (SEQ IDNO: 928), PIP-72Fh (SEQ ID NO: 932), PIP-72Fi (SEQ ID NO: 933), PIP-72Fj(SEQ ID NO: 934), PIP-72Fk (SEQ ID NO: 935), PIP-72FI (SEQ ID NO: 936),PIP-72Gg (SEQ ID NO: 939), PIP-72Gh (SEQ ID NO: 940), PIP-72Gi (SEQ IDNO: 941), PIP-72Gk (SEQ ID NO: 943), PIP-72GI (SEQ ID NO: 944), PIP-72Gm(SEQ ID NO: 945) or PIP-72Gn (SEQ ID NO: 946) in any combination.

It is recognized that DNA sequences may be altered by various methods,and that these alterations may result in DNA sequences encoding proteinswith amino acid sequences different than that encoded by the wild-type(or native) pesticidal protein. In some embodiments a PIP-72 polypeptidemay be altered in various ways including amino acid substitutions,deletions, truncations and insertions of one or more amino acids,including up to 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45 or more amino acid substitutions,deletions and/or insertions or combinations thereof compared to SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ IDNO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, anyone of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ IDNO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, SEQ ID NO: 846,SEQ ID NO: 847, SEQ ID NO: 848, SEQ ID NO: 849, any one of SEQ ID NO:903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO:939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQID NO: 945, or SEQ ID NO: 946.

Methods for such manipulations are generally known in the art. Forexample, amino acid sequence variants of a PIP-72 polypeptide can beprepared by mutations in the DNA. This may also be accomplished by oneof several forms of mutagenesis and/or in directed evolution. In someaspects, the changes encoded in the amino acid sequence will notsubstantially affect the function of the protein. Such variants willpossess the desired pesticidal activity. However, it is understood thatthe ability of a PIP-72 polypeptide to confer pesticidal activity may beimproved by the use of such techniques upon the compositions of thisdisclosure.

For example, conservative amino acid substitutions may be made at one ormore, predicted, nonessential amino acid residues. A “nonessential”amino acid residue is a residue that can be altered from the wild-typesequence of a PIP-72 polypeptide without altering the biologicalactivity. A “conservative amino acid substitution” is one in which theamino acid residue is replaced with an amino acid residue having asimilar side chain. Families of amino acid residues having similar sidechains have been defined in the art. These families include: amino acidswith basic side chains (e.g., lysine, arginine, histidine); acidic sidechains (e.g., aspartic acid, glutamic acid); polar, negatively chargedresidues and their amides (e.g., aspartic acid, asparagine, glutamic,acid, glutamine; uncharged polar side chains (e.g., glycine, asparagine,glutamine, serine, threonine, tyrosine, cysteine); small aliphatic,nonpolar or slightly polar residues (e.g., Alanine, serine, threonine,proline, glycine); nonpolar side chains (e.g., alanine, valine, leucine,isoleucine, proline, phenylalanine, methionine, tryptophan); largealiphatic, nonpolar residues (e.g., methionine, leucine, isoleucine,valine, cystine); beta-branched side chains (e.g., threonine, valine,isoleucine); aromatic side chains (e.g., tyrosine, phenylalanine,tryptophan, histidine); large aromatic side chains (e.g., tyrosine,phenylalanine, tryptophan).

Amino acid substitutions may be made in nonconserved regions that retainfunction. In general, such substitutions would not be made for conservedamino acid residues or for amino acid residues residing within aconserved motif, where such residues are essential for protein activity.Examples of residues that are conserved and that may be essential forprotein activity include, for example, residues that are identicalbetween all proteins contained in an alignment of similar or relatedtoxins to the sequences of the embodiments (e.g., residues that areidentical in an alignment of homologs). Examples of residues that areconserved but that may allow conservative amino acid substitutions andstill retain activity include, for example, residues that have onlyconservative substitutions between all proteins contained in analignment of similar or related toxins to the sequences of theembodiments (e.g., residues that have only conservative substitutionsbetween all proteins contained in the alignment of the homologs).However, one of skill in the art would understand that functionalvariants may have minor conserved or nonconserved alterations in theconserved residues. Guidance as to appropriate amino acid substitutionsthat do not affect biological activity of the protein of interest may befound in the model of Dayhoff, et al., (1978) Atlas of Protein Sequenceand Structure (Natl. Biomed. Res. Found., Washington, D.C.), hereinincorporated by reference.

In making such changes, the hydropathic index of amino acids may beconsidered. The importance of the hydropathic amino acid index inconferring interactive biologic function on a protein is generallyunderstood in the art (Kyte and Doolittle, (1982) J Mol Biol.157(1):105-32). It is accepted that the relative hydropathic characterof the amino acid contributes to the secondary structure of theresultant protein, which in turn defines the interaction of the proteinwith other molecules, for example, enzymes, substrates, receptors, DNA,antibodies, antigens, and the like.

It is known in the art that certain amino acids may be substituted byother amino acids having a similar hydropathic index or score and stillresult in a protein with similar biological activity, i.e., still obtaina biological functionally equivalent protein. Each amino acid has beenassigned a hydropathic index on the basis of its hydrophobicity andcharge characteristics (Kyte and Doolittle, ibid). These are: isoleucine(+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8);cysteine/cystine (+2.5); methionine (+1.9); alanine (+1.8); glycine(−0.4); threonine (−0.7); serine (−0.8); tryptophan (−0.9); tyrosine(−1.3); proline (−1.6); histidine (−3.2); glutamate (−3.5); glutamine(−3.5); aspartate (−3.5); asparagine (−3.5); lysine (−3.9) and arginine(−4.5). In making such changes, the substitution of amino acids whosehydropathic indices are within +2 is preferred, those which are within+1 are particularly preferred, and those within +0.5 are even moreparticularly preferred.

It is also understood in the art that the substitution of like aminoacids can be made effectively on the basis of hydrophilicity. U.S. Pat.No. 4,554,101, states that the greatest local average hydrophilicity ofa protein, as governed by the hydrophilicity of its adjacent aminoacids, correlates with a biological property of the protein.

As detailed in U.S. Pat. No. 4,554,101, the following hydrophilicityvalues have been assigned to amino acid residues: arginine (+3.0);lysine (+3.0); aspartate (+3.0.+0.1); glutamate (+3.0.+0.1); serine(+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine(−0.4); proline (−0.5.+0.1); alanine (−0.5); histidine (−0.5); cysteine(−1.0); methionine (−1.3); valine (−1.5); leucine (−1.8); isoleucine(−1.8); tyrosine (−2.3); phenylalanine (−2.5); tryptophan (−3.4).

Alternatively, alterations may be made to the protein sequence of manyproteins at the amino or carboxy terminus without substantiallyaffecting activity. This can include insertions, deletions oralterations introduced by modern molecular methods, such as PCR,including PCR amplifications that alter or extend the protein codingsequence by virtue of inclusion of amino acid encoding sequences in theoligonucleotides utilized in the PCR amplification. Alternatively, theprotein sequences added can include entire protein-coding sequences,such as those used commonly in the art to generate protein fusions. Suchfusion proteins are often used to (1) increase expression of a proteinof interest (2) introduce a binding domain, enzymatic activity orepitope to facilitate either protein purification, protein detection orother experimental uses known in the art (3) target secretion ortranslation of a protein to a subcellular organelle, such as theperiplasmic space of Gram-negative bacteria, mitochondria orchloroplasts of plants or the endoplasmic reticulum of eukaryotic cells,the latter of which often results in glycosylation of the protein.

Variant nucleotide and amino acid sequences of the disclosure alsoencompass sequences derived from mutagenic and recombinogenic proceduressuch as DNA shuffling. With such a procedure, one or more differentPIP-72 polypeptide coding regions can be used to create a new PIP-72polypeptide possessing the desired properties. In this manner, librariesof recombinant polynucleotides are generated from a population ofrelated sequence polynucleotides comprising sequence regions that havesubstantial sequence identity and can be homologously recombined invitro or in vivo. For example, using this approach, sequence motifsencoding a domain of interest may be shuffled between a pesticidal geneand other known pesticidal genes to obtain a new gene coding for aprotein with an improved property of interest, such as an increasedinsecticidal activity. Strategies for such DNA shuffling are known inthe art. See, for example, Stemmer, (1994) Proc. Natl. Acad. Sci. USA91:10747-10751; Stemmer, (1994) Nature 370:389-391; Crameri, et al.,(1997) Nature Biotech. 15:436-438; Moore, et al., (1997) J. Mol. Biol.272:336-347; Zhang, et al., (1997) Proc. Natl. Acad. Sci. USA94:4504-4509; Crameri, et al., (1998) Nature 391:288-291; and U.S. Pat.Nos. 5,605,793 and 5,837,458.

Domain swapping or shuffling is another mechanism for generating alteredPIP-72 polypeptides. Domains may be swapped between PIP-72 polypeptides,resulting in hybrid or chimeric toxins with improved insecticidalactivity or target spectrum. Methods for generating recombinant proteinsand testing them for pesticidal activity are well known in the art (see,for example, Naimov, et al., (2001) Appl. Environ. Microbiol.67:5328-5330; de Maagd, et al., (1996) Appl. Environ. Microbiol.62:1537-1543; Ge, et al., (1991) J. Biol. Chem. 266:17954-17958;Schnepf, et al., (1990) J. Biol. Chem. 265:20923-20930; Rang, et al.,91999) Appl. Environ. Microbiol. 65:2918-2925).

Both DNA shuffling and site-directed mutagenesis were used to definepolypeptide sequences that possess pesticidal activity. In Examples 8 &9 DNA shuffling was used to generate a library of active variants byrecombination of the diversity present in GBP_A3175 (SEQ ID NO: 20) andPIP-72 Da (SEQ ID NO: 10). The person skilled in the art will be able touse comparisons to other proteins or functional assays to further definemotifs. High throughput screening can be used to test variations ofthose motifs to determine the role of specific residues. Given thatknowledge for several motifs, one can then define the requirements for afunctional protein. Knowledge of the motifs allows the skilled artisanto design sequence variations that would not impact function.

Alignment of homologs of PIP-72 homologs (FIGS. 1, 2, 3, 4 & 5) allowedidentification of residues that are conserved among homologs in thisfamily (FIG. 1). In Example 10 and 11, saturation mutagenesis was usedto make and test substitutions at selected amino acid positions. Thesemutants were tested for activity and a number of active substitutionsnot present among the homologues were identified providing anunderstanding of the functional constraints at these residues.

In some embodiments polypeptides are provided comprising an amino acidsequence having at least 75%, at least 80%, at least 85%, at least 90%,at least 95% or greater sequence identity to the amino acid sequence setforth in SEQ ID NO: 20, SEQ ID NO: 24 SEQ ID NO: 30, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO:937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQ ID NO: 948,wherein the polypeptide has insecticidal activity.

Compositions

Compositions comprising a PIP-72 polypeptide are also embraced. In someembodiments the composition comprises a PIP-72 polypeptide. In someembodiments the composition comprises a PIP-72 fusion protein.

Antibodies

Antibodies to a PIP-72 polypeptide of the embodiments or to variants orfragments thereof are also encompassed. The antibodies of the disclosureinclude polyclonal and monoclonal antibodies as well as fragmentsthereof which retain their ability to bind to PIP-72 proteins found inthe insect gut. An antibody, monoclonal antibody or fragment thereof issaid to be capable of binding a molecule if it is capable ofspecifically reacting with the molecule to thereby bind the molecule tothe antibody, monoclonal antibody or fragment thereof. The term“antibody” (Ab) or “monoclonal antibody” (Mab) is meant to includeintact molecules as well as fragments or binding regions or domainsthereof (such as, for example, Fab and F(ab).sub.2 fragments) which arecapable of binding hapten. Such fragments are typically produced byproteolytic cleavage, such as papain or pepsin. Alternatively,hapten-binding fragments can be produced through the application ofrecombinant DNA technology or through synthetic chemistry. Methods forthe preparation of the antibodies of the present disclosure aregenerally known in the art. For example, see, Antibodies, A LaboratoryManual, Ed Harlow and David Lane (eds.) Cold Spring Harbor Laboratory,N.Y. (1988), as well as the references cited therein. Standard referenceworks setting forth the general principles of immunology include: Klein,J. Immunology: The Science of Cell-Noncell Discrimination, John Wiley &Sons, N.Y. (1982); Dennett, et al., Monoclonal Antibodies, Hybridoma: ANew Dimension in Biological Analyses, Plenum Press, N.Y. (1980) andCampbell, “Monoclonal Antibody Technology,” In Laboratory Techniques inBiochemistry and Molecular Biology, Vol. 13, Burdon, et al., (eds.),Elsevier, Amsterdam (1984). See also, U.S. Pat. Nos. 4,196,265;4,609,893; 4,713,325; 4,714,681; 4,716,111; 4,716,117 and 4,720,459.PIP-72 polypeptide polypeptide antibodies or antigen-binding portionsthereof can be produced by a variety of techniques, includingconventional monoclonal antibody methodology, for example the standardsomatic cell hybridization technique of Kohler and Milstein, (1975)Nature 256:495. Other techniques for producing monoclonal antibody canalso be employed such as viral or oncogenic transformation of Blymphocytes. An animal system for preparing hybridomas is a murinesystem. Immunization protocols and techniques for isolation of immunizedsplenocytes for fusion are known in the art. Fusion partners (e.g.,murine myeloma cells) and fusion procedures are also known. The antibodyand monoclonal antibodies of the disclosure can be prepared by utilizinga PIP-72 polypeptide polypeptide as antigens.

A kit for detecting the presence of a PIP-72 polypeptide polypeptide ordetecting the presence of a nucleotide sequence encoding a PIP-72polypeptide polypeptide, in a sample is provided. In one embodiment, thekit provides antibody-based reagents for detecting the presence of aPIP-72 polypeptide polypeptide in a tissue sample. In anotherembodiment, the kit provides labeled nucleic acid probes useful fordetecting the presence of one or more polynucleotides encoding PIP-72polypeptide(s). The kit is provided along with appropriate reagents andcontrols for carrying out a detection method, as well as instructionsfor use of the kit.

Receptor Identification and Isolation

Receptors to the PIP-72 polypeptide of the embodiments or to variants orfragments thereof, are also encompassed. Methods for identifyingreceptors are well known in the art (see, Hofmann, et. al., (1988) Eur.J. Biochem. 173:85-91; Gill, et al., (1995) J. Biol. Chem. 27277-27282)can be employed to identify and isolate the receptor that recognizes thePIP-72 polypeptides using the brush-border membrane vesicles fromsusceptible insects. In addition to the radioactive labeling methodlisted in the cited literatures, PIP-72 polypeptide can be labeled withfluorescent dye and other common labels such as streptavidin.Brush-border membrane vesicles (BBMV) of susceptible insects such assoybean looper and stink bugs can be prepared according to the protocolslisted in the references and separated on SDS-PAGE gel and blotted onsuitable membrane. Labeled PIP-72 polypeptides can be incubated withblotted membrane of BBMV and labeled the PIP-72 polypeptides can beidentified with the labeled reporters. Identification of protein band(s)that interact with the PIP-72 polypeptides can be detected by N-terminalamino acid gas phase sequencing or mass spectrometry based proteinidentification method (Patterson, (1998) 10.22, 1-24, Current Protocolin Molecular Biology published by John Wiley & Son Inc). Once theprotein is identified, the corresponding gene can be cloned from genomicDNA or cDNA library of the susceptible insects and binding affinity canbe measured directly with the PIP-72 polypeptides. Receptor function forinsecticidal activity by the PIP-72 polypeptides can be verified byaccomplished by RNAi type of gene knock out method (Rajagopal, et al.,(2002) J. Biol. Chem. 277:46849-46851).

Nucleotide Constructs, Expression Cassettes and Vectors

The use of the term “nucleotide constructs” herein is not intended tolimit the embodiments to nucleotide constructs comprising DNA. Those ofordinary skill in the art will recognize that nucleotide constructsparticularly polynucleotides and oligonucleotides composed ofribonucleotides and combinations of ribonucleotides anddeoxyribonucleotides may also be employed in the methods disclosedherein. The nucleotide constructs, nucleic acids, and nucleotidesequences of the embodiments additionally encompass all complementaryforms of such constructs, molecules, and sequences. Further, thenucleotide constructs, nucleotide molecules, and nucleotide sequences ofthe embodiments encompass all nucleotide constructs, molecules, andsequences which can be employed in the methods of the embodiments fortransforming plants including, but not limited to, those comprised ofdeoxyribonucleotides, ribonucleotides, and combinations thereof. Suchdeoxyribonucleotides and ribonucleotides include both naturallyoccurring molecules and synthetic analogues. The nucleotide constructs,nucleic acids, and nucleotide sequences of the embodiments alsoencompass all forms of nucleotide constructs including, but not limitedto, single-stranded forms, double-stranded forms, hairpins,stem-and-loop structures and the like.

A further embodiment relates to a transformed organism such as anorganism selected from plant and insect cells, bacteria, yeast,baculovirus, protozoa, nematodes and algae. The transformed organismcomprises a DNA molecule of the embodiments, an expression cassettecomprising the DNA molecule or a vector comprising the expressioncassette, which may be stably incorporated into the genome of thetransformed organism.

The sequences of the embodiments are provided in DNA constructs forexpression in the organism of interest. The construct will include 5′and 3′ regulatory sequences operably linked to a sequence of theembodiments. The term “operably linked” as used herein refers to afunctional linkage between a promoter and a second sequence, wherein thepromoter sequence initiates and mediates transcription of the DNAsequence corresponding to the second sequence. Generally, operablylinked means that the nucleic acid sequences being linked are contiguousand where necessary to join two protein coding regions in the samereading frame. The construct may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple DNA constructs.

In some embodiments the DNA construct comprises a polynucleotideencoding a PIP-72 polypeptide of the embodiments operably linked to aheterologous regulatory sequence.

In some embodiments the DNA construct comprises a polynucleotideencoding a polypeptide having at least 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% orgreater sequence identity to the amino acid sequence of SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQ IDNO: 948, operably linked to a heterologous regulatory sequence.

In some embodiments the DNA construct comprises a polynucleotideencoding a polypeptide comprising amino acid sequence of SEQ ID NO: 20,SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26, SEQ ID NO: 30, SEQ ID NO:34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQ IDNO: 948 operably linked to a heterologous regulatory sequence.

Such a DNA construct is provided with a plurality of restriction sitesfor insertion of the PIP-72 polypeptide gene sequence to be under thetranscriptional regulation of the regulatory regions. The DNA constructmay additionally contain selectable marker genes.

The DNA construct will generally include in the 5′ to 3′ direction oftranscription: a transcriptional and translational initiation region(i.e., a promoter), a DNA sequence of the embodiments, and atranscriptional and translational termination region (i.e., terminationregion) functional in the organism serving as a host. Thetranscriptional initiation region (i.e., the promoter) may be native,analogous, foreign or heterologous to the host organism and/or to thesequence of the embodiments. Additionally, the promoter may be thenatural sequence or alternatively a synthetic sequence. The term“foreign” as used herein indicates that the promoter is not found in thenative organism into which the promoter is introduced. Where thepromoter is “foreign” or “heterologous” to the sequence of theembodiments, it is intended that the promoter is not the native ornaturally occurring promoter for the operably linked sequence of theembodiments. As used herein, a chimeric gene comprises a coding sequenceoperably linked to a transcription initiation region that isheterologous to the coding sequence. Where the promoter is a native ornatural sequence, the expression of the operably linked sequence isaltered from the wild-type expression, which results in an alteration inphenotype.

In some embodiments the DNA construct may also include a transcriptionalenhancer sequence. As used herein, the term an “enhancer” refers to aDNA sequence which can stimulate promoter activity, and may be an innateelement of the promoter or a heterologous element inserted to enhancethe level or tissue-specificity of a promoter. Various enhancers areknown in the art including for example, introns with gene expressionenhancing properties in plants (US Patent Application Publication Number2009/0144863, the ubiquitin intron (i.e., the maize ubiquitin intron 1(see, for example, NCBI sequence S94464; Christensen and Quail (1996)Transgenic Res. 5:213-218; Christensen et al. (1992) Plant MolecularBiology 18:675-689)), the omega enhancer or the omega prime enhancer(Gallie, et al., (1989) Molecular Biology of RNA ed. Cech (Liss, NewYork) 237-256 and Gallie, et al., (1987) Gene 60:217-25), the CaMV 35Senhancer (see, e.g., Benfey, et al., (1990) EMBO J. 9:1685-96), themaize Adh1 intron (Kyozuka et al. (1991) Mol. Gen. Genet. 228:40-48;Kyozuka et al. (1990) Maydica 35:353-357), the enhancers of U.S. Pat.No. 7,803,992, and the sugarcane bacilliform viral (SCBV) enhancer ofWO2013130813 may also be used, each of which is incorporated byreference. The above list of transcriptional enhancers is not meant tobe limiting. Any appropriate transcriptional enhancer can be used in theembodiments.

The termination region may be native with the transcriptional initiationregion, may be native with the operably linked DNA sequence of interest,may be native with the plant host or may be derived from another source(i.e., foreign or heterologous to the promoter, the sequence ofinterest, the plant host or any combination thereof).

Convenient termination regions are available from the Ti-plasmid of A.tumefaciens, such as the octopine synthase and nopaline synthasetermination regions. See also, Guerineau, et al., (1991) Mol. Gen.Genet. 262:141-144; Proudfoot, (1991) Cell 64:671-674; Sanfacon, et al.,(1991) Genes Dev. 5:141-149; Mogen, et al., (1990) Plant Cell2:1261-1272; Munroe, et al., (1990) Gene 91:151-158; Ballas, et al.,(1989) Nucleic Acids Res. 17:7891-7903 and Joshi, et al., (1987) NucleicAcid Res. 15:9627-9639.

Where appropriate, a nucleic acid may be optimized for increasedexpression in the host organism. Thus, where the host organism is aplant, the synthetic nucleic acids can be synthesized usingplant-preferred codons for improved expression. See, for example,Campbell and Gowri, (1990) Plant Physiol. 92:1-11 for a discussion ofhost-preferred codon usage. For example, although nucleic acid sequencesof the embodiments may be expressed in both monocotyledonous anddicotyledonous plant species, sequences can be modified to account forthe specific codon preferences and GC content preferences ofmonocotyledons or dicotyledons as these preferences have been shown todiffer (Murray et al. (1989) Nucleic Acids Res. 17:477-498). Thus, themaize-preferred codon for a particular amino acid may be derived fromknown gene sequences from maize. Maize codon usage for 28 genes frommaize plants is listed in Table 4 of Murray, et al., supra. Methods areavailable in the art for synthesizing plant-preferred genes. See, forexample, U.S. Pat. Nos. 5,380,831, and 5,436,391 and Murray, et al.,(1989) Nucleic Acids Res. 17:477-498, and Liu H et al. Mol Bio Rep37:677-684, 2010, herein incorporated by reference. A Zea maize codonusage table can be also found atkazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=4577, which can beaccessed using the www prefix. Table 2 shows a maize optimal codonanalysis (adapted from Liu H et al. Mol Bio Rep 37:677-684, 2010).

TABLE 2 Amino High Low Amino High Low Acid Codon Count RSCU Count RSCUAcid Codon Count RSCU Count RSCU Phe UUU 115 0.04 2,301 1.22 Ala GCU 6290.17 3,063 1.59 UUC* 5,269 1.96 1,485 0.78 GCC* 8,057 2.16 1,136 0.59Ser UCU 176 0.13 2,498 1.48 GCA 369 0.1 2,872 1.49 UCC* 3,489 2.48 1,0740.63 GCG* 5,835 1.57 630 0.33 UCA 104 0.07 2,610 1.54 Tyr UAU 71 0.041,632 1.22 UCG* 1,975 1.4 670 0.4 UAC* 3,841 1.96 1,041 0.78 AGU 77 0.051,788 1.06 His CAU 131 0.09 1,902 1.36 AGC* 2,617 1.86 1,514 0.89 CAC*2,800 1.91 897 0.64 Leu UUA 10 0.01 1,326 0.79 Cys UGU 52 0.04 1,2331.12 UUG 174 0.09 2,306 1.37 UGC* 2,291 1.96 963 0.88 CUU 223 0.11 2,3961.43 Gln CAA 99 0.05 2,312 1.04 CUC* 5,979 3.08 1,109 0.66 CAG* 3,5571.95 2,130 0.96 CUA 106 0.05 1,280 0.76 Arg CGU 153 0.12 751 0.74 CUG*5,161 2.66 1,646 0.98 CGC* 4,278 3.25 466 0.46 Pro CCU 427 0.22 1,9001.47 CGA 92 0.07 659 0.65 CCC* 3,035 1.59 601 0.47 CGG* 1,793 1.36 6310.62 CCA 311 0.16 2,140 1.66 AGA 83 0.06 1,948 1.91 CCG* 3,846 2.02 5130.4 AGG* 1,493 1.14 1,652 1.62 Ile AUU 138 0.09 2,388 1.3 Asn AAU 1310.07 3,074 1.26 AUC* 4,380 2.85 1,353 0.74 AAC* 3,814 1.93 1,807 0.74AUA 88 0.06 1,756 0.96 Lys AAA 130 0.05 3,215 0.98 Thr ACU 136 0.091,990 1.43 AAG* 5,047 1.95 3,340 1.02 ACC* 3,398 2.25 991 0.71 Asp GAU312 0.09 4,217 1.38 ACA 133 0.09 2,075 1.5 GAC* 6,729 1.91 1,891 0.62ACG* 2,378 1.57 495 0.36 Gly GGU 363 0.13 2,301 1.35 Val GUU 182 0.072,595 1.51 GGC* 7,842 2.91 1,282 0.75 GUC* 4,584 1.82 1,096 0.64 GGA 3970.15 2,044 1.19 GUA 74 0.03 1,325 0.77 GGG* 2,186 0.81 1,215 0.71 GUG*5,257 2.08 1,842 1.07 Glu GAA 193 0.06 4,080 1.1 GAG* 6,010 1.94 3,3070.9 Codon usage was compared using Chi squared contingency test toidentify optimal codons. Codons that occur significantly more often(P\0.01) are indicated with an asterisk.

A Glycine max codon usage table is shown in Table 3 and can also befound at kazusa.or.jp/codon/cgi-bin/showcodon.cgi?species=3847&aa=1&style=N, which can be accessed using the www prefix.

TABLE 3 TTT F 21.2 (10493) TCT S 18.4 (9107) TTC F 21.2 (10487) TCC S12.9 (6409) TTA L 9.2 (4545) TCA S 15.6 (7712) TTG L 22.9 (11340) TCG S4.8 (2397) CTT L 23.9 (11829) CCT P 18.9 (9358) CTC L 17.1 (8479) CCC P10.1 (5010) CTA L 8.5 (4216) CCA P 19.1 (9461) CTG L 12.7 (6304) CCG P4.7 (2312) ATT I 25.1 (12411) ACT T 17.1 (8490) ATC I 16.3 (8071) ACC T14.3 (7100) ATA I 12.9 (6386) ACA T 14.9 (7391) ATG M 22.7 (11218) ACG T4.3 (2147) GTT V 26.1 (12911) GCT A 26.7 (13201) GTC V 11.9 (5894) GCC A16.2 (8026) GTA V 7.7 (3803) GCA A 21.4 (10577) GTG V 21.4 (10610) GCG A6.3 (3123) TAT Y 15.7 (7779) TGT C 8.1 (3995) TAC Y 14.9 (7367) TGC C8.0 (3980) TAA * 0.9 (463) TGA * 1.0 (480) TAG * 0.5 (263) TGG W 13.0(6412) CAT H 14.0 (6930) CGT R 6.6 (3291) CAC H 11.6 (5759) CGC R 6.2(3093) CAA Q 20.5 (10162) CGA R 4.1 (2018) CAG Q 16.2 (8038) CGG R 3.1(1510) AAT N 22.4 (11088) AGT S 12.6 (6237) AAC N 22.8 (11284) AGC S11.3 (5594) AAA K 26.9 (13334) AGA R 14.8 (7337) AAG K 35.9 (17797) AGGR 13.3 (6574) GAT D 32.4 (16040) GGT G 20.9 (10353) GAC D 20.4 (10097)GGC G 13.4 (6650) GAA E 33.2 (16438) GGA G 22.3 (11022) GAG E 33.2(16426) GGG G 13.0 (6431)

In some embodiments the recombinant nucleic acid molecule encoding aPIP-72 polypeptide has maize optimized codons.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other well-characterized sequences that maybe deleterious to gene expression. The GC content of the sequence may beadjusted to levels average for a given cellular host, as calculated byreference to known genes expressed in the host cell. The term “hostcell” as used herein refers to a cell which contains a vector andsupports the replication and/or expression of the expression vector isintended. Host cells may be prokaryotic cells such as E. coli oreukaryotic cells such as yeast, insect, amphibian or mammalian cells ormonocotyledonous or dicotyledonous plant cells. An example of amonocotyledonous host cell is a maize host cell. When possible, thesequence is modified to avoid predicted hairpin secondary mRNAstructures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein, et al., (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallie,et al., (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus), human immunoglobulin heavy-chain binding protein (BiP) (Macejak,et al., (1991) Nature 353:90-94); untranslated leader from the coatprotein mRNA of alfalfa mosaic virus (AMV RNA 4) (Jobling, et al.,(1987) Nature 325:622-625); tobacco mosaic virus leader (TMV) (Gallie,et al., (1989) in Molecular Biology of RNA, ed. Cech (Liss, New York),pp. 237-256) and maize chlorotic mottle virus leader (MCMV) (Lommel, etal., (1991) Virology 81:382-385). See also, Della-Cioppa, et al., (1987)Plant Physiol. 84:965-968. Such constructs may also contain a “signalsequence” or “leader sequence” to facilitate co-translational orpost-translational transport of the peptide to certain intracellularstructures such as the chloroplast (or other plastid), endoplasmicreticulum or Golgi apparatus.

“Signal sequence” as used herein refers to a sequence that is known orsuspected to result in cotranslational or post-translational peptidetransport across the cell membrane. In eukaryotes, this typicallyinvolves secretion into the Golgi apparatus, with some resultingglycosylation. Insecticidal toxins of bacteria are often synthesized asprotoxins, which are protolytically activated in the gut of the targetpest (Chang, (1987) Methods Enzymol. 153:507-516). In some embodiments,the signal sequence is located in the native sequence or may be derivedfrom a sequence of the embodiments. “Leader sequence” as used hereinrefers to any sequence that when translated, results in an amino acidsequence sufficient to trigger co-translational transport of the peptidechain to a subcellular organelle. Thus, this includes leader sequencestargeting transport and/or glycosylation by passage into the endoplasmicreticulum, passage to vacuoles, plastids including chloroplasts,mitochondria, and the like. Nuclear-encoded proteins targeted to thechloroplast thylakoid lumen compartment have a characteristic bipartitetransit peptide, composed of a stromal targeting signal peptide and alumen targeting signal peptide. The stromal targeting information is inthe amino-proximal portion of the transit peptide. The lumen targetingsignal peptide is in the carboxyl-proximal portion of the transitpeptide, and contains all the information for targeting to the lumen.Recent research in proteomics of the higher plant chloroplast hasachieved in the identification of numerous nuclear-encoded lumenproteins (Kieselbach et al. FEBS LETT 480:271-276, 2000; Peltier et al.Plant Cell 12:319-341, 2000; Bricker et al. Biochim. Biophys Acta1503:350-356, 2001), the lumen targeting signal peptide of which canpotentially be used in accordance with the present disclosure. About 80proteins from Arabidopsis, as well as homologous proteins from spinachand garden pea, are reported by Kieselbach et al., PhotosynthesisResearch, 78:249-264, 2003. In particular, Table 2 of this publication,which is incorporated into the description herewith by reference,discloses 85 proteins from the chloroplast lumen, identified by theiraccession number (see also US Patent Application Publication2009/09044298). In addition, the recently published draft version of therice genome (Goff et al, Science 296:92-100, 2002) is a suitable sourcefor lumen targeting signal peptide which may be used in accordance withthe present disclosure.

Suitable chloroplast transit peptides (CTP) are well known to oneskilled in the art also include chimeric CTPs comprising but not limitedto, an N-terminal domain, a central domain or a C-terminal domain from aCTP from Oryza sativa 1-deoxy-D xyulose-5-Phosphate Synthase Oryzasativa-Superoxide dismutase Oryza sativa-soluble starch synthase Oryzasativa-NADP-dependent Malic acid enzyme Oryzasativa-Phospho-2-dehydro-3-deoxyheptonate Aldolase 2 Oryzasativa-L-Ascorbate peroxidase 5 Oryza sativa-Phosphoglucan waterdikinase, Zea Mays ssRUBISCO, Zea Mays-beta-glucosidase, Zea Mays-Malatedehydrogenase, Zea Mays Thioredoxin M-type (US Patent ApplicationPublication 2012/0304336). Chloroplast transit peptides of US PatentPublications US20130205440A1, US20130205441A1 and US20130210114A1.

The PIP-72 polypeptide gene to be targeted to the chloroplast may beoptimized for expression in the chloroplast to account for differencesin codon usage between the plant nucleus and this organelle. In thismanner, the nucleic acids of interest may be synthesized usingchloroplast-preferred codons. See, for example, U.S. Pat. No. 5,380,831,herein incorporated by reference.

In preparing the expression cassette, the various DNA fragments may bemanipulated so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used in the practice of the embodiments.The promoters can be selected based on the desired outcome. The nucleicacids can be combined with constitutive, tissue-preferred, inducible orother promoters for expression in the host organism. Promoters of thepresent invention include homologues of cis elements known to effectgene regulation that show homology with the promoter sequences of thepresent invention. These cis elements include, but are not limited to,oxygen responsive cis elements (Cowen et al., J Biol. Chem.268(36):26904-26910 (1993)), light regulatory elements (Bruce andQuaill, Plant Cell 2 (11):1081-1089 (1990); Bruce et al., EMBO J.10:3015-3024 (1991); Rocholl et al., Plant Sci. 97:189-198 (1994); Blocket al., Proc. Natl. Acad. Sci. USA 87:5387-5391 (1990); Giuliano et al.,Proc. Natl. Acad. Sci. USA 85:7089-7093 (1988); Staiger et al., Proc.Natl. Acad. Sci. USA 86:6930-6934 (1989); Izawa et al., Plant Cell6:1277-1287 (1994); Menkens et al., Trends in Biochemistry 20:506-510(1995); Foster et al., FASEB J. 8:192-200 (1994); Plesse et al., Mol GenGene 254:258-266 (1997); Green et al., EMBO J. 6:2543-2549 (1987);Kuhlemeier et al., Ann. Rev Plant Physiol. 38:221-257 (1987); Villain etal., J. Biol. Chem. 271:32593-32598 (1996); Lam et al., Plant Cell2:857-866 (1990); Gilmartin et al., Plant Cell 2:369-378 (1990); Dattaet al., Plant Cell 1:1069-1077 (1989); Gilmartin et al., Plant Cell2:369-378 (1990); Castresana et al., EMBO J. 7:1929-1936 (1988); Ueda etal., Plant Cell 1:217-227 (1989); Terzaghi et al., Annu. Rev. PlantPhysiol. Plant Mol. Biol. 46:445-474 (1995); Green et al., EMBO J.6:2543-2549 (1987); Villain et al., J. Biol. Chem. 271:32593-32598(1996); Tjaden et al., Plant Cell 6:107-118 (1994); Tjaden et al., PlantPhysiol. 108:1109-1117 (1995); Ngai et al., Plant J. 12:1021-1234(1997); Bruce et al., EMBO J. 10:3015-3024 (1991); Ngai et al., Plant J.12:1021-1034 (1997)), elements responsive to gibberellin, (Muller etal., J. Plant Physiol. 145:606-613 (1995); Croissant et al., PlantScience 116:27-35 (1996); Lohmer et al., EMBO J. 10:617-624 (1991);Rogers et al., Plant Cell 4:1443-1451 (1992); Lanahan et al., Plant Cell4:203-211 (1992); Skriver et al., Proc. Natl. Acad. Sci. USA88:7266-7270 (1991); Gilmartin et al., Plant Cell 2:369-378 (1990);Huang et al., Plant Mol. Biol. 14:655-668 (1990), Gubler et al., PlantCell 7:1879-1891 (1995)), elements responsive to abscisic acid, (Busk etal., Plant Cell 9:2261-2270 (1997); Guiltinan et al., Science250:267-270 (1990); Shen et al., Plant Cell 7:295-307 (1995); Shen etal., Plant Cell 8:1107-1119 (1996); Seo et al., Plant Mol. Biol.27:1119-1131 (1995); Marcotte et al., Plant Cell 1:969-976 (1989); Shenet al., Plant Cell 7:295-307 (1995); Iwasaki et al., Mol Gen Genet247:391-398 (1995); Hattori et al., Genes Dev. 6:609-618 (1992); Thomaset al., Plant Cell 5:1401-1410 (1993)), elements similar to abscisicacid responsive elements, (Ellerstrom et al., Plant Mol. Biol.32:1019-1027 (1996)), auxin responsive elements (Liu et al., Plant Cell6:645-657 (1994); Liu et al., Plant Physiol. 115:397-407 (1997); Kosugiet al., Plant J. 7:877-886 (1995); Kosugi et al., Plant Cell 9:1607-1619(1997); Ballas et al., J. Mol. Biol. 233:580-596 (1993)), a cis elementresponsive to methyl jasmonate treatment (Beaudoin and Rothstein, PlantMol. Biol. 33:835-846 (1997)), a cis element responsive to abscisic acidand stress response (Straub et al., Plant Mol. Biol. 26:617-630 (1994)),ethylene responsive cis elements (Itzhaki et al., Proc. Natl. Acad. Sci.USA 91:8925-8929 (1994); Montgomery et al., Proc. Natl. Acad. Sci. USA90:5939-5943 (1993); Sessa et al., Plant Mol. Biol. 28:145-153 (1995);Shinshi et al., Plant Mol. Biol. 27:923-932 (1995)), salicylic acid cisresponsive elements, (Strange et al., Plant J. 11:1315-1324 (1997); Qinet al., Plant Cell 6:863-874 (1994)), a cis element that responds towater stress and abscisic acid (Lam et al., J. Biol. Chem.266:17131-17135 (1991); Thomas et al., Plant Cell 5:1401-1410 (1993);Pla et al., Plant Mol Biol 21:259-266 (1993)), a cis element essentialfor M phase-specific expression (Ito et al., Plant Cell 10:331-341(1998)), sucrose responsive elements (Huang et al., Plant Mol. Biol.14:655-668 (1990); Hwang et al., Plant Mol Biol 36:331-341 (1998);Grierson et al., Plant J. 5:815-826 (1994)), heat shock responseelements (Pelham et al., Trends Genet. 1:31-35 (1985)), elementsresponsive to auxin and/or salicylic acid and also reported for lightregulation (Lam et al., Proc. Natl. Acad. Sci. USA 86:7890-7897 (1989);Benfey et al., Science 250:959-966 (1990)), elements responsive toethylene and salicylic acid (Ohme-Takagi et al., Plant Mol. Biol.15:941-946 (1990)), elements responsive to wounding and abiotic stress(Loake et al., Proc. Natl. Acad. Sci. USA 89:9230-9234 (1992); Mhiri etal., Plant Mol. Biol. 33:257-266 (1997)), antioxidant response elements(Rushmore et al., J. Biol. Chem. 266:11632-11639; Dalton et al., NucleicAcids Res. 22:5016-5023 (1994)), Sph elements (Suzuki et al., Plant Cell9:799-807 1997)), elicitor responsive elements, (Fukuda et al., PlantMol. Biol. 34:81-87 (1997); Rushton et al., EMBO J. 15:5690-5700(1996)), metal responsive elements (Stuart et al., Nature 317:828-831(1985); Westin et al., EMBO J. 7:3763-3770 (1988); Thiele et al.,Nucleic Acids Res. 20:1183-1191 (1992); Faisst et al., Nucleic AcidsRes. 20:3-26 (1992)), low temperature responsive elements, (Baker etal., Plant Mol. Biol. 24:701-713 (1994); Jiang et al., Plant Mol. Biol.30:679-684 (1996); Nordin et al., Plant Mol. Biol. 21:641-653 (1993);Zhou et al., J. Biol. Chem. 267:23515-23519 (1992)), drought responsiveelements, (Yamaguchi et al., Plant Cell 6:251-264 (1994); Wang et al.,Plant Mol. Biol. 28:605-617 (1995); Bray E A, Trends in Plant Science2:48-54 (1997)) enhancer elements for glutenin, (Colot et al., EMBO J.6:3559-3564 (1987); Thomas et al., Plant Cell 2:1171-1180 (1990); Kreiset al., Philos. Trans. R. Soc. Lond., B314:355-365 (1986)),light-independent regulatory elements, (Lagrange et al., Plant Cell9:1469-1479 (1997); Villain et al., J. Biol. Chem. 271:32593-32598(1996)), OCS enhancer elements, (Bouchez et al., EMBO J. 8:4197-4204(1989); Foley et al., Plant J. 3:669-679 (1993)), ACGT elements, (Fosteret al., FASEB J. 8:192-200 (1994); Izawa et al., Plant Cell 6:1277-1287(1994); Izawa et al., J. Mol. Biol. 230:1131-1144 (1993)), negative ciselements in plastid related genes, (Zhou et al., J. Biol. Chem.267:23515-23519 (1992); Lagrange et al., Mol. Cell Biol. 13:2614-2622(1993); Lagrange et al., Plant Cell 9:1469-1479 (1997); Zhou et al., J.Biol. Chem. 267:23515-23519 (1992)), prolamin box elements, (Forde etal., Nucleic Acids Res. 13:7327-7339 (1985); Colot et al., EMBO J.6:3559-3564 (1987); Thomas et al., Plant Cell 2:1171-1180 (1990);Thompson et al., Plant Mol. Biol. 15:755-764 (1990); Vicente et al.,Proc. Natl. Acad. Sci. USA 94:7685-7690 (1997)), elements in enhancersfrom the IgM heavy chain gene (Gillies et al., Cell 33:717-728 (1983);Whittier et al., Nucleic Acids Res. 15:2515-2535 (1987)). Examples ofpromoters include: those described in U.S. Pat. No. 6,437,217 (maizeRS81 promoter), U.S. Pat. No. 5,641,876 (rice actin promoter), U.S. Pat.No. 6,426,446 (maize RS324 promoter), U.S. Pat. No. 6,429,362 (maizePR-1 promoter), U.S. Pat. No. 6,232,526 (maize A3 promoter), U.S. Pat.No. 6,177,611 (constitutive maize promoters), U.S. Pat. Nos. 5,322,938,5,352,605, 5,359,142 and 5,530,196 (35S promoter), U.S. Pat. No.6,433,252 (maize L3 oleosin promoter, P-Zm.L3), U.S. Pat. No. 6,429,357(rice actin 2 promoter as well as a rice actin 2 intron), U.S. Pat. No.5,837,848 (root specific promoter), U.S. Pat. No. 6,294,714 (lightinducible promoters), U.S. Pat. No. 6,140,078 (salt induciblepromoters), U.S. Pat. No. 6,252,138 (pathogen inducible promoters), U.S.Pat. No. 6,175,060 (phosphorus deficiency inducible promoters), U.S.Pat. No. 6,635,806 (gama-coixin promoter, P-CI.Gcx), U.S. patentapplication Ser. No. 09/757,089 (maize chloroplast aldolase promoter),and U.S. Pat. No. 8,772,466 (maize transcription factor Nuclear Factor B(NFB2)).

Suitable constitutive promoters for use in a plant host cell include,for example, the core promoter of the Rsyn7 promoter and otherconstitutive promoters disclosed in WO 1999/43838 and U.S. Pat. No.6,072,050; the core CaMV 35S promoter (Odell, et al., (1985) Nature313:810-812); rice actin (McElroy, et al., (1990) Plant Cell 2:163-171);ubiquitin (Christensen, et al., (1989) Plant Mol. Biol. 12:619-632 andChristensen, et al., (1992) Plant Mol. Biol. 18:675-689); pEMU (Last, etal., (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten, et al., (1984)EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026) and thelike. Other constitutive promoters include, for example, those discussedin U.S. Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142 and 6,177,611. Suitable constitutivepromoters also include promoters that have strong expression in nearlyall tissues but have low expression in pollen, including but not limitedto: Banana Streak Virus (Acuminata Yunnan) promoters (BSV(AY)) disclosedin US patent U.S. Pat. No. 8,338,662; Banana Streak Virus (AcuminataVietnam) promoters (BSV(AV)) disclosed in US patent U.S. Pat. No.8,350,121; and Banana Streak Virus (Mysore) promoters (BSV(MYS))disclosed in US patent U.S. Pat. No. 8,395,022.

Depending on the desired outcome, it may be beneficial to express thegene from an inducible promoter. Of particular interest for regulatingthe expression of the nucleotide sequences of the embodiments in plantsare wound-inducible promoters. Such wound-inducible promoters, mayrespond to damage caused by insect feeding, and include potatoproteinase inhibitor (pin II) gene (Ryan, (1990) Ann. Rev. Phytopath.28:425-449; Duan, et al., (1996) Nature Biotechnology 14:494-498); wun1and wun2, U.S. Pat. No. 5,428,148; win1 and win2 (Stanford, et al.,(1989) Mol. Gen. Genet. 215:200-208); systemin (McGurl, et al., (1992)Science 225:1570-1573); WIP1 (Rohmeier, et al., (1993) Plant Mol. Biol.22:783-792; Eckelkamp, et al., (1993) FEBS Letters 323:73-76); MPI gene(Corderok, et al., (1994) Plant J. 6(2):141-150) and the like, hereinincorporated by reference.

Additionally, pathogen-inducible promoters may be employed in themethods and nucleotide constructs of the embodiments. Suchpathogen-inducible promoters include those from pathogenesis-relatedproteins (PR proteins), which are induced following infection by apathogen; e.g., PR proteins, SAR proteins, beta-1,3-glucanase,chitinase, etc. See, for example, Redolfi, et al., (1983) Neth. J. PlantPathol. 89:245-254; Uknes, et al., (1992) Plant Cell 4: 645-656 and VanLoon, (1985) Plant Mol. Virol. 4:111-116. See also, WO 1999/43819,herein incorporated by reference.

Of interest are promoters that are expressed locally at or near the siteof pathogen infection. See, for example, Marineau, et al., (1987) PlantMol. Biol. 9:335-342; Matton, et al., (1989) Molecular Plant-MicrobeInteractions 2:325-331; Somsisch, et al., (1986) Proc. Natl. Acad. Sci.USA 83:2427-2430; Somsisch, et al., (1988) Mol. Gen. Genet. 2:93-98 andYang, (1996) Proc. Natl. Acad. Sci. USA 93:14972-14977. See also, Chen,et al., (1996) Plant J. 10:955-966; Zhang, et al., (1994) Proc. Natl.Acad. Sci. USA 91:2507-2511; Warner, et al., (1993) Plant J. 3:191-201;Siebertz, et al., (1989) Plant Cell 1:961-968; U.S. Pat. No. 5,750,386(nematode-inducible) and the references cited therein. Of particularinterest is the inducible promoter for the maize PRms gene, whoseexpression is induced by the pathogen Fusarium moniliforme (see, forexample, Cordero, et al., (1992) Physiol. Mol. Plant Path. 41:189-200).

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression or a chemical-repressible promoter, where application ofthe chemical represses gene expression. Chemical-inducible promoters areknown in the art and include, but are not limited to, the maize In2-2promoter, which is activated by benzenesulfonamide herbicide safeners,the maize GST promoter, which is activated by hydrophobic electrophiliccompounds that are used as pre-emergent herbicides, and the tobaccoPR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters (see, for example, the glucocorticoid-inducible promoter inSchena, et al., (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis, et al., (1998) Plant J. 14(2):247-257) andtetracycline-inducible and tetracycline-repressible promoters (see, forexample, Gatz, et al., (1991) Mol. Gen. Genet. 227:229-237 and U.S. Pat.Nos. 5,814,618 and 5,789,156), herein incorporated by reference.

Tissue-preferred promoters can be utilized to target enhanced PIP-72polypeptide expression within a particular plant tissue.Tissue-preferred promoters include those discussed in Yamamoto, et al.,(1997) Plant J. 12(2)255-265; Kawamata, et al., (1997) Plant CellPhysiol. 38(7):792-803; Hansen, et al., (1997) Mol. Gen Genet.254(3):337-343; Russell, et al., (1997) Transgenic Res. 6(2):157-168;Rinehart, et al., (1996) Plant Physiol. 112(3):1331-1341; Van Camp, etal., (1996) Plant Physiol. 112(2):525-535; Canevascini, et al., (1996)Plant Physiol. 112(2):513-524; Yamamoto, et al., (1994) Plant CellPhysiol. 35(5):773-778; Lam, (1994) Results Probl. Cell Differ.20:181-196; Orozco, et al., (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka, et al., (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590 andGuevara-Garcia, et al., (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto, et al., (1997) Plant J. 12(2):255-265; Kwon, et al., (1994)Plant Physiol. 105:357-67; Yamamoto, et al., (1994) Plant Cell Physiol.35(5):773-778; Gotor, et al., (1993) Plant J. 3:509-18; Orozco, et al.,(1993) Plant Mol. Biol. 23(6):1129-1138 and Matsuoka, et al., (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Root-preferred or root-specific promoters are known and can be selectedfrom the many available from the literature or isolated de novo fromvarious compatible species. See, for example, Hire, et al., (1992) PlantMol. Biol. 20(2):207-218 (soybean root-specific glutamine synthetasegene); Keller and Baumgartner, (1991) Plant Cell 3(10):1051-1061(root-specific control element in the GRP 1.8 gene of French bean);Sanger, et al., (1990) Plant Mol. Biol. 14(3):433-443 (root-specificpromoter of the mannopine synthase (MAS) gene of Agrobacteriumtumefaciens) and Miao, et al., (1991) Plant Cell 3(1):11-22 (full-lengthcDNA clone encoding cytosolic glutamine synthetase (GS), which isexpressed in roots and root nodules of soybean). See also, Bogusz, etal., (1990) Plant Cell 2(7):633-641, where two root-specific promotersisolated from hemoglobin genes from the nitrogen-fixing nonlegumeParasponia andersonii and the related non-nitrogen-fixing nonlegumeTrema tomentosa are described. The promoters of these genes were linkedto a β-glucuronidase reporter gene and introduced into both thenonlegume Nicotiana tabacum and the legume Lotus corniculatus, and inboth instances root-specific promoter activity was preserved. Leach andAoyagi, (1991) describe their analysis of the promoters of the highlyexpressed rolC and rolD root-inducing genes of Agrobacterium rhizogenes(see, Plant Science (Limerick) 79(1):69-76). They concluded thatenhancer and tissue-preferred DNA determinants are dissociated in thosepromoters. Teeri, et al., (1989) used gene fusion to lacZ to show thatthe Agrobacterium T-DNA gene encoding octopine synthase is especiallyactive in the epidermis of the root tip and that the TR2′ gene is rootspecific in the intact plant and stimulated by wounding in leaf tissue,an especially desirable combination of characteristics for use with aninsecticidal or larvicidal gene (see, EMBO J. 8(2):343-350). The TR1′gene fused to nptll (neomycin phosphotransferase II) showed similarcharacteristics. Additional root-preferred promoters include theVfENOD-GRP3 gene promoter (Kuster, et al., (1995) Plant Mol. Biol.29(4):759-772) and rolB promoter (Capana, et al., (1994) Plant Mol.Biol. 25(4):681-691. See also, U.S. Pat. Nos. 5,837,876; 5,750,386;5,633,363; 5,459,252; 5,401,836; 5,110,732 and 5,023,179. Arabidopsisthaliana root-preferred regulatory sequences are disclosed in US PatentApplication US20130117883. Root-preferred sorghum (Sorghum bicolor) RCc3promoters are disclosed in US Patent Application US20120210463. Theroot-preferred maize promoters of US Patent Application Publication20030131377, U.S. Pat. Nos. 7,645,919, and 8,735,655. The rootcap-specific 1 (ZmRCP1) maize promoters of US Patent ApplicationPublication 20130025000. The root-preferred maize promoters of US PatentApplication Publication 20130312136.

“Seed-preferred” promoters include both “seed-specific” promoters (thosepromoters active during seed development such as promoters of seedstorage proteins) as well as “seed-germinating” promoters (thosepromoters active during seed germination). See, Thompson, et al., (1989)BioEssays 10:108, herein incorporated by reference. Such seed-preferredpromoters include, but are not limited to, Cim1 (cytokinin-inducedmessage); cZ19B1 (maize 19 kDa zein); and milps(myo-inositol-1-phosphate synthase) (see, U.S. Pat. No. 6,225,529,herein incorporated by reference). Gamma-zein and Glb-1 areendosperm-specific promoters. For dicots, seed-specific promotersinclude, but are not limited to, Kunitz trypsin inhibitor 3 (KTi3)(Jofuku and Goldberg, (1989) Plant Cell 1:1079-1093), bean β-phaseolin,napin, β-conglycinin, glycinin 1, soybean lectin, cruciferin, and thelike. For monocots, seed-specific promoters include, but are not limitedto, maize 15 kDa zein, 22 kDa zein, 27 kDa zein, g-zein, waxy, shrunken1, shrunken 2, globulin 1, etc. See also, WO 2000/12733, whereseed-preferred promoters from end1 and end2 genes are disclosed; hereinincorporated by reference. In dicots, seed specific promoters includebut are not limited to seed coat promoter from Arabidopsis, pBAN; andthe early seed promoters from Arabidopsis, p26, p63, and p63tr (U.S.Pat. Nos. 7,294,760 and 7,847,153). A promoter that has “preferred”expression in a particular tissue is expressed in that tissue to agreater degree than in at least one other plant tissue. Sometissue-preferred promoters show expression almost exclusively in theparticular tissue.

Where low level expression is desired, weak promoters will be used.Generally, the term “weak promoter” as used herein refers to a promoterthat drives expression of a coding sequence at a low level. By low levelexpression at levels of about 1/1000 transcripts to about 1/100,000transcripts to about 1/500,000 transcripts is intended. Alternatively,it is recognized that the term “weak promoters” also encompassespromoters that drive expression in only a few cells and not in others togive a total low level of expression. Where a promoter drives expressionat unacceptably high levels, portions of the promoter sequence can bedeleted or modified to decrease expression levels.

Such weak constitutive promoters include, for example the core promoterof the Rsyn7 promoter (WO 1999/43838 and U.S. Pat. No. 6,072,050), thecore 35S CaMV promoter, and the like. Other constitutive promotersinclude, for example, those disclosed in U.S. Pat. Nos. 5,608,149;5,608,144; 5,604,121; 5,569,597; 5,466,785; 5,399,680; 5,268,463;5,608,142 and 6,177,611, herein incorporated by reference.

The above list of promoters is not meant to be limiting. Any appropriatepromoter can be used in the embodiments.

Generally, the expression cassette will comprise a selectable markergene for the selection of transformed cells. Selectable marker genes areutilized for the selection of transformed cells or tissues. Marker genesinclude genes encoding antibiotic resistance, such as those encodingneomycin phosphotransferase II (NEO) and hygromycin phosphotransferase(HPT), as well as genes conferring resistance to herbicidal compounds,such as glufosinate ammonium, bromoxynil, imidazolinones and2,4-dichlorophenoxyacetate (2,4-D). Additional examples of suitableselectable marker genes include, but are not limited to, genes encodingresistance to chloramphenicol (Herrera Estrella, et al., (1983) EMBO J.2:987-992); methotrexate (Herrera Estrella, et al., (1983) Nature303:209-213 and Meijer, et al., (1991) Plant Mol. Biol. 16:807-820);streptomycin (Jones, et al., (1987) Mol. Gen. Genet. 210:86-91);spectinomycin (Bretagne-Sagnard, et al., (1996) Transgenic Res.5:131-137); bleomycin (Hille, et al., (1990) Plant Mol. Biol.7:171-176); sulfonamide (Guerineau, et al., (1990) Plant Mol. Biol.15:127-136); bromoxynil (Stalker, et al., (1988) Science 242:419-423);glyphosate (Shaw, et al., (1986) Science 233:478-481 and U.S. patentapplication Ser. Nos. 10/004,357 and 10/427,692); phosphinothricin(DeBlock, et al., (1987) EMBO J. 6:2513-2518). See generally, Yarranton,(1992) Curr. Opin. Biotech. 3:506-511; Christopherson, et al., (1992)Proc. Natl. Acad. Sci. USA 89:6314-6318; Yao, et al., (1992) Cell71:63-72; Reznikoff, (1992) Mol. Microbio. 6:2419-2422; Barkley, et al.,(1980) in The Operon, pp. 177-220; Hu, et al., (1987) Cell 48:555-566;Brown, et al., (1987) Cell 49:603-612; Figge, et al., (1988) Cell52:713-722; Deuschle, et al., (1989) Proc. Natl. Acad. Sci. USA86:5400-5404; Fuerst, et al., (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle, et al., (1990) Science 248:480-483; Gossen,(1993) Ph.D. Thesis, University of Heidelberg; Reines, et al., (1993)Proc. Natl. Acad. Sci. USA 90:1917-1921; Labow, et al., (1990) Mol.Cell. Biol. 10:3343-3356; Zambretti, et al., (1992) Proc. Natl. Acad.Sci. USA 89:3952-3956; Baim, et al., (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski, et al., (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman, (1989) Topics Mol. Struc. Biol. 10:143-162;Degenkolb, et al., (1991) Antimicrob. Agents Chemother. 35:1591-1595;Kleinschnidt, et al., (1988) Biochemistry 27:1094-1104; Bonin, (1993)Ph.D. Thesis, University of Heidelberg; Gossen, et al., (1992) Proc.Natl. Acad. Sci. USA 89:5547-5551; Oliva, et al., (1992) Antimicrob.Agents Chemother. 36:913-919; Hlavka, et al., (1985) Handbook ofExperimental Pharmacology, Vol. 78 (Springer-Verlag, Berlin) and Gill,et al., (1988) Nature 334:721-724. Such disclosures are hereinincorporated by reference.

The above list of selectable marker genes is not meant to be limiting.Any selectable marker gene can be used in the embodiments.

Plant Transformation

The methods of the embodiments involve introducing a polypeptide orpolynucleotide into a plant. “Introducing” is as used herein meanspresenting to the plant the polynucleotide or polypeptide in such amanner that the sequence gains access to the interior of a cell of theplant. The methods of the embodiments do not depend on a particularmethod for introducing a polynucleotide or polypeptide into a plant,only that the polynucleotide or polypeptides gains access to theinterior of at least one cell of the plant. Methods for introducingpolynucleotide or polypeptides into plants are known in the artincluding, but not limited to, stable transformation methods, transienttransformation methods, and virus-mediated methods.

“Stable transformation” is as used herein means that the nucleotideconstruct introduced into a plant integrates into the genome of theplant and is capable of being inherited by the progeny thereof.“Transient transformation” as used herein means that a polynucleotide isintroduced into the plant and does not integrate into the genome of theplant or a polypeptide is introduced into a plant. “Plant” as usedherein refers to whole plants, plant organs (e.g., leaves, stems, roots,etc.), seeds, plant cells, propagules, embryos and progeny of the same.Plant cells can be differentiated or undifferentiated (e.g. callus,suspension culture cells, protoplasts, leaf cells, root cells, phloemcells and pollen).

Transformation protocols as well as protocols for introducing nucleotidesequences into plants may vary depending on the type of plant or plantcell, i.e., monocot or dicot, targeted for transformation. Suitablemethods of introducing nucleotide sequences into plant cells andsubsequent insertion into the plant genome include microinjection(Crossway, et al., (1986) Biotechniques 4:320-334), electroporation(Riggs, et al., (1986) Proc. Natl. Acad. Sci. USA 83:5602-5606),Agrobacterium-mediated transformation (U.S. Pat. Nos. 5,563,055 and5,981,840), direct gene transfer (Paszkowski, et al., (1984) EMBO J.3:2717-2722) and ballistic particle acceleration (see, for example, U.S.Pat. Nos. 4,945,050; 5,879,918; 5,886,244 and 5,932,782; Tomes, et al.,(1995) in Plant Cell, Tissue, and Organ Culture: Fundamental Methods,ed. Gamborg and Phillips, (Springer-Verlag, Berlin) and McCabe, et al.,(1988) Biotechnology 6:923-926) and Led transformation (WO 00/28058).For potato transformation see, Tu, et al., (1998) Plant MolecularBiology 37:829-838 and Chong, et al., (2000) Transgenic Research9:71-78. Additional transformation procedures can be found inWeissinger, et al., (1988) Ann. Rev. Genet. 22:421-477; Sanford, et al.,(1987) Particulate Science and Technology 5:27-37 (onion); Christou, etal., (1988) Plant Physiol. 87:671-674 (soybean); McCabe, et al., (1988)Bio/Technology 6:923-926 (soybean); Finer and McMullen, (1991) In VitroCell Dev. Biol. 27P:175-182 (soybean); Singh, et al., (1998) Theor.Appl. Genet. 96:319-324 (soybean); Datta, et al., (1990) Biotechnology8:736-740 (rice); Klein, et al., (1988) Proc. Natl. Acad. Sci. USA85:4305-4309 (maize); Klein, et al., (1988) Biotechnology 6:559-563(maize); U.S. Pat. Nos. 5,240,855; 5,322,783 and 5,324,646; Klein, etal., (1988) Plant Physiol. 91:440-444 (maize); Fromm, et al., (1990)Biotechnology 8:833-839 (maize); Hooykaas-Van Slogteren, et al., (1984)Nature (London) 311:763-764; U.S. Pat. No. 5,736,369 (cereals);Bytebier, et al., (1987) Proc. Natl. Acad. Sci. USA 84:5345-5349(Liliaceae); De Wet, et al., (1985) in The Experimental Manipulation ofOvule Tissues, ed. Chapman, et al., (Longman, N.Y.), pp. 197-209(pollen); Kaeppler, et al., (1990) Plant Cell Reports 9:415-418 andKaeppler, et al., (1992) Theor. Appl. Genet. 84:560-566(whisker-mediated transformation); D'Halluin, et al., (1992) Plant Cell4:1495-1505 (electroporation); Li, et al., (1993) Plant Cell Reports12:250-255 and Christou and Ford, (1995) Annals of Botany 75:407-413(rice); Osjoda, et al., (1996) Nature Biotechnology 14:745-750 (maizevia Agrobacterium tumefaciens); all of which are herein incorporated byreference.

In specific embodiments, the sequences of the embodiments can beprovided to a plant using a variety of transient transformation methods.Such transient transformation methods include, but are not limited to,the introduction of the PIP-72 polypeptide or variants and fragmentsthereof directly into the plant or the introduction of the PIP-72polypeptide transcript into the plant. Such methods include, forexample, microinjection or particle bombardment. See, for example,Crossway, et al., (1986) Mol Gen. Genet. 202:179-185; Nomura, et al.,(1986) Plant Sci. 44:53-58; Hepler, et al., (1994) Proc. Natl. Acad.Sci. 91:2176-2180 and Hush, et al., (1994) The Journal of Cell Science107:775-784, all of which are herein incorporated by reference.Alternatively, the PIP-72 polypeptide polynucleotide can be transientlytransformed into the plant using techniques known in the art. Suchtechniques include viral vector system and the precipitation of thepolynucleotide in a manner that precludes subsequent release of the DNA.Thus, transcription from the particle-bound DNA can occur, but thefrequency with which it is released to become integrated into the genomeis greatly reduced. Such methods include the use of particles coatedwith polyethylimine (PEI; Sigma #P3143).

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO 1999/25855and WO 1999/25853, all of which are herein incorporated by reference.Briefly, the polynucleotide of the embodiments can be contained intransfer cassette flanked by two non-identical recombination sites. Thetransfer cassette is introduced into a plant have stably incorporatedinto its genome a target site which is flanked by two non-identicalrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome.

Plant transformation vectors may be comprised of one or more DNA vectorsneeded for achieving plant transformation. For example, it is a commonpractice in the art to utilize plant transformation vectors that arecomprised of more than one contiguous DNA segment. These vectors areoften referred to in the art as “binary vectors”. Binary vectors as wellas vectors with helper plasmids are most often used forAgrobacterium-mediated transformation, where the size and complexity ofDNA segments needed to achieve efficient transformation is quite large,and it is advantageous to separate functions onto separate DNAmolecules. Binary vectors typically contain a plasmid vector thatcontains the cis-acting sequences required for T-DNA transfer (such asleft border and right border), a selectable marker that is engineered tobe capable of expression in a plant cell, and a “gene of interest” (agene engineered to be capable of expression in a plant cell for whichgeneration of transgenic plants is desired). Also present on thisplasmid vector are sequences required for bacterial replication. Thecis-acting sequences are arranged in a fashion to allow efficienttransfer into plant cells and expression therein. For example, theselectable marker gene and the pesticidal gene are located between theleft and right borders. Often a second plasmid vector contains thetrans-acting factors that mediate T-DNA transfer from Agrobacterium toplant cells. This plasmid often contains the virulence functions (Virgenes) that allow infection of plant cells by Agrobacterium, andtransfer of DNA by cleavage at border sequences and vir-mediated DNAtransfer, as is understood in the art (Hellens and Mullineaux, (2000)Trends in Plant Science 5:446-451). Several types of Agrobacteriumstrains (e.g. LBA4404, GV3101, EHA101, EHA105, etc.) can be used forplant transformation. The second plasmid vector is not necessary fortransforming the plants by other methods such as microprojection,microinjection, electroporation, polyethylene glycol, etc.

In general, plant transformation methods involve transferringheterologous DNA into target plant cells (e.g., immature or matureembryos, suspension cultures, undifferentiated callus, protoplasts,etc.), followed by applying a maximum threshold level of appropriateselection (depending on the selectable marker gene) to recover thetransformed plant cells from a group of untransformed cell mass.Following integration of heterologous foreign DNA into plant cells, onethen applies a maximum threshold level of appropriate selection in themedium to kill the untransformed cells and separate and proliferate theputatively transformed cells that survive from this selection treatmentby transferring regularly to a fresh medium. By continuous passage andchallenge with appropriate selection, one identifies and proliferatesthe cells that are transformed with the plasmid vector. Molecular andbiochemical methods can then be used to confirm the presence of theintegrated heterologous gene of interest into the genome of thetransgenic plant.

Explants are typically transferred to a fresh supply of the same mediumand cultured routinely. Subsequently, the transformed cells aredifferentiated into shoots after placing on regeneration mediumsupplemented with a maximum threshold level of selecting agent. Theshoots are then transferred to a selective rooting medium for recoveringrooted shoot or plantlet. The transgenic plantlet then grows into amature plant and produces fertile seeds (e.g., Hiei, et al., (1994) ThePlant Journal 6:271-282; Ishida, et al., (1996) Nature Biotechnology14:745-750). Explants are typically transferred to a fresh supply of thesame medium and cultured routinely. A general description of thetechniques and methods for generating transgenic plants are found inAyres and Park, (1994) Critical Reviews in Plant Science 13:219-239 andBommineni and Jauhar, (1997) Maydica 42:107-120. Since the transformedmaterial contains many cells; both transformed and non-transformed cellsare present in any piece of subjected target callus or tissue or groupof cells. The ability to kill non-transformed cells and allowtransformed cells to proliferate results in transformed plant cultures.Often, the ability to remove non-transformed cells is a limitation torapid recovery of transformed plant cells and successful generation oftransgenic plants.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick, et al.,(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting hybrid having constitutive or inducible expression ofthe desired phenotypic characteristic identified. Two or moregenerations may be grown to ensure that expression of the desiredphenotypic characteristic is stably maintained and inherited and thenseeds harvested to ensure that expression of the desired phenotypiccharacteristic has been achieved.

The nucleotide sequences of the embodiments may be provided to the plantby contacting the plant with a virus or viral nucleic acids. Generally,such methods involve incorporating the nucleotide construct of interestwithin a viral DNA or RNA molecule. It is recognized that therecombinant proteins of the embodiments may be initially synthesized aspart of a viral polyprotein, which later may be processed by proteolysisin vivo or in vitro to produce the desired PIP-72 polypeptide. It isalso recognized that such a viral polyprotein, comprising at least aportion of the amino acid sequence of a PIP-72 polypeptide of theembodiments, may have the desired pesticidal activity. Such viralpolyproteins and the nucleotide sequences that encode for them areencompassed by the embodiments. Methods for providing plants withnucleotide constructs and producing the encoded proteins in the plants,which involve viral DNA or RNA molecules are known in the art. See, forexample, U.S. Pat. Nos. 5,889,191; 5,889,190; 5,866,785; 5,589,367 and5,316,931; herein incorporated by reference.

Methods for transformation of chloroplasts are known in the art. See,for example, Svab, et al., (1990) Proc. Natl. Acad. Sci. USA87:8526-8530; Svab and Maliga, (1993) Proc. Natl. Acad. Sci. USA90:913-917; Svab and Maliga, (1993) EMBO J. 12:601-606. The methodrelies on particle gun delivery of DNA containing a selectable markerand targeting of the DNA to the plastid genome through homologousrecombination. Additionally, plastid transformation can be accomplishedby transactivation of a silent plastid-borne transgene bytissue-preferred expression of a nuclear-encoded and plastid-directedRNA polymerase. Such a system has been reported in McBride, et al.,(1994) Proc. Natl. Acad. Sci. USA 91:7301-7305.

The embodiments further relate to plant-propagating material of atransformed plant of the embodiments including, but not limited to,seeds, tubers, corms, bulbs, leaves and cuttings of roots and shoots.

The embodiments may be used for transformation of any plant species,including, but not limited to, monocots and dicots. Examples of plantsof interest include, but are not limited to, corn (Zea mays), Brassicasp. (e.g., B. napus, B. rapa, B. juncea), particularly those Brassicaspecies useful as sources of seed oil, alfalfa (Medicago sativa), rice(Oryza sativa), rye (Secale cereale), sorghum (Sorghum bicolor, Sorghumvulgare), millet (e.g., pearl millet (Pennisetum glaucum), proso millet(Panicum miliaceum), foxtail millet (Setaria italica), finger millet(Eleusine coracana)), sunflower (Helianthus annuus), safflower(Carthamus tinctorius), wheat (Triticum aestivum), soybean (Glycinemax), tobacco (Nicotiana tabacum), potato (Solanum tuberosum), peanuts(Arachis hypogaea), cotton (Gossypium barbadense, Gossypium hirsutum),sweet potato (Ipomoea batatus), cassava (Manihot esculenta), coffee(Coffea spp.), coconut (Cocos nucifera), pineapple (Ananas comosus),citrus trees (Citrus spp.), cocoa (Theobroma cacao), tea (Camelliasinensis), banana (Musa spp.), avocado (Persea americana), fig (Ficuscasica), guava (Psidium guajava), mango (Mangifera indica), olive (Oleaeuropaea), papaya (Carica papaya), cashew (Anacardium occidentale),macadamia (Macadamia integrifolia), almond (Prunus amygdalus), sugarbeets (Beta vulgaris), sugarcane (Saccharum spp.), oats, barley,vegetables ornamentals, and conifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum. Conifers that may beemployed in practicing the embodiments include, for example, pines suchas loblolly pine (Pinus taeda), slash pine (Pinus elliotil), ponderosapine (Pinus ponderosa), lodgepole pine (Pinus contorta), and Montereypine (Pinus radiata); Douglas-fir (Pseudotsuga menziesii); Westernhemlock (Tsuga canadensis); Sitka spruce (Picea glauca); redwood(Sequoia sempervirens); true firs such as silver fir (Abies amabilis)and balsam fir (Abies balsamea); and cedars such as Western red cedar(Thuja plicata) and Alaska yellow-cedar (Chamaecyparis nootkatensis).Plants of the embodiments include crop plants (for example, corn,alfalfa, sunflower, Brassica, soybean, cotton, safflower, peanut,sorghum, wheat, millet, tobacco, etc.), such as corn and soybean plants.

Turf grasses include, but are not limited to: annual bluegrass (Poaannua); annual ryegrass (Lolium multiflorum); Canada bluegrass (Poacompressa); Chewing's fescue (Festuca rubra); colonial bentgrass(Agrostis tenuis); creeping bentgrass (Agrostis palustris); crestedwheatgrass (Agropyron desertorum); fairway wheatgrass (Agropyroncristatum); hard fescue (Festuca longifolia); Kentucky bluegrass (Poapratensis); orchardgrass (Dactylis glomerata); perennial ryegrass(Lolium perenne); red fescue (Festuca rubra); redtop (Agrostis alba);rough bluegrass (Poa trivialis); sheep fescue (Festuca ovina); smoothbromegrass (Bromus inermis); tall fescue (Festuca arundinacea); timothy(Phleum pratense); velvet bentgrass (Agrostis canina); weepingalkaligrass (Puccinellia distans); western wheatgrass (Agropyronsmithi); Bermuda grass (Cynodon spp.); St. Augustine grass (Stenotaphrumsecundatum); zoysia grass (Zoysia spp.); Bahia grass (Paspalum notatum);carpet grass (Axonopus affinis); centipede grass (Eremochloaophiuroides); kikuyu grass (Pennisetum clandesinum); seashore paspalum(Paspalum vaginatum); blue gramma (Bouteloua gracilis); buffalo grass(Buchloe dactyloids); sideoats gramma (Bouteloua curtipendula).

Plants of interest include grain plants that provide seeds of interest,oil-seed plants, and leguminous plants. Seeds of interest include grainseeds, such as corn, wheat, barley, rice, sorghum, rye, millet, etc.Oil-seed plants include cotton, soybean, safflower, sunflower, Brassica,maize, alfalfa, palm, coconut, flax, castor, olive, etc. Leguminousplants include beans and peas. Beans include guar, locust bean,fenugreek, soybean, garden beans, cowpea, mungbean, lima bean, favabean, lentils, chickpea, etc.

Evaluation of Plant Transformation

Following introduction of heterologous foreign DNA into plant cells, thetransformation or integration of heterologous gene in the plant genomeis confirmed by various methods such as analysis of nucleic acids,proteins and metabolites associated with the integrated gene.

PCR analysis is a rapid method to screen transformed cells, tissue orshoots for the presence of incorporated gene at the earlier stage beforetransplanting into the soil (Sambrook and Russell, (2001) MolecularCloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y.). PCR is carried out using oligonucleotide primersspecific to the gene of interest or Agrobacterium vector background,etc.

Plant transformation may be confirmed by Southern blot analysis ofgenomic DNA (Sambrook and Russell, (2001) supra). In general, total DNAis extracted from the transformant, digested with appropriaterestriction enzymes, fractionated in an agarose gel and transferred to anitrocellulose or nylon membrane. The membrane or “blot” is then probedwith, for example, radiolabeled 32P target DNA fragment to confirm theintegration of introduced gene into the plant genome according tostandard techniques (Sambrook and Russell, (2001) supra).

In Northern blot analysis, RNA is isolated from specific tissues oftransformant, fractionated in a formaldehyde agarose gel, and blottedonto a nylon filter according to standard procedures that are routinelyused in the art (Sambrook and Russell, (2001) supra). Expression of RNAencoded by the pesticidal gene is then tested by hybridizing the filterto a radioactive probe derived from a pesticidal gene, by methods knownin the art (Sambrook and Russell, (2001) supra).

Western blot, biochemical assays and the like may be carried out on thetransgenic plants to confirm the presence of protein encoded by thepesticidal gene by standard procedures (Sambrook and Russell, 2001,supra) using antibodies that bind to one or more epitopes present on thePIP-72 polypeptide.

Stacking of Traits in Transgenic Plant

Transgenic plants may comprise a stack of one or more insecticidalpolynucleotides disclosed herein with one or more additionalpolynucleotides resulting in the production or suppression of multiplepolypeptide sequences. Transgenic plants comprising stacks ofpolynucleotide sequences can be obtained by either or both oftraditional breeding methods or through genetic engineering methods.These methods include, but are not limited to, breeding individual lineseach comprising a polynucleotide of interest, transforming a transgenicplant comprising a gene disclosed herein with a subsequent gene andco-transformation of genes into a single plant cell. As used herein, theterm “stacked” includes having the multiple traits present in the sameplant (i.e., both traits are incorporated into the nuclear genome, onetrait is incorporated into the nuclear genome and one trait isincorporated into the genome of a plastid or both traits areincorporated into the genome of a plastid). In one non-limiting example,“stacked traits” comprise a molecular stack where the sequences arephysically adjacent to each other. A trait, as used herein, refers tothe phenotype derived from a particular sequence or groups of sequences.Co-transformation of genes can be carried out using singletransformation vectors comprising multiple genes or genes carriedseparately on multiple vectors. If the sequences are stacked bygenetically transforming the plants, the polynucleotide sequences ofinterest can be combined at any time and in any order. The traits can beintroduced simultaneously in a co-transformation protocol with thepolynucleotides of interest provided by any combination oftransformation cassettes. For example, if two sequences will beintroduced, the two sequences can be contained in separatetransformation cassettes (trans) or contained on the same transformationcassette (cis). Expression of the sequences can be driven by the samepromoter or by different promoters. In certain cases, it may bedesirable to introduce a transformation cassette that will suppress theexpression of the polynucleotide of interest. This may be combined withany combination of other suppression cassettes or overexpressioncassettes to generate the desired combination of traits in the plant. Itis further recognized that polynucleotide sequences can be stacked at adesired genomic location using a site-specific recombination system.See, for example, WO 1999/25821, WO 1999/25854, WO 1999/25840, WO1999/25855 and WO 1999/25853, all of which are herein incorporated byreference.

In some embodiments the polynucleotides encoding the PIP-72 polypeptidesdisclosed herein, alone or stacked with one or more additional insectresistance traits can be stacked with one or more additional inputtraits (e.g., herbicide resistance, fungal resistance, virus resistance,stress tolerance, disease resistance, male sterility, stalk strength,and the like) or output traits (e.g., increased yield, modifiedstarches, improved oil profile, balanced amino acids, high lysine ormethionine, increased digestibility, improved fiber quality, droughtresistance, and the like). Thus, the polynucleotide embodiments can beused to provide a complete agronomic package of improved crop qualitywith the ability to flexibly and cost effectively control any number ofagronomic pests.

Transgenes useful for stacking include but are not limited to:

1. Transgenes that Confer Resistance to Insects or Disease and thatEncode:

(A) plant disease resistance genes. Plant defenses are often activatedby specific interaction between the product of a disease resistance gene(R) in the plant and the product of a corresponding avirulence (Avr)gene in the pathogen. A plant variety can be transformed with clonedresistance gene to engineer plants that are resistant to specificpathogen strains. See, for example, Jones, et al., (1994) Science266:789 (cloning of the tomato Cf-9 gene for resistance to Cladosporiumfulvum); Martin, et al., (1993) Science 262:1432 (tomato Pto gene forresistance to Pseudomonas syringae pv. tomato encodes a protein kinase);Mindrinos, et al., (1994) Cell 78:1089 (Arabidopsis RSP2 gene forresistance to Pseudomonas syringae), McDowell and Woffenden, (2003)Trends Biotechnol. 21(4):178-83 and Toyoda, et al., (2002) TransgenicRes. 11(6):567-82. A plant resistant to a disease is one that is moreresistant to a pathogen as compared to the wild type plant.

(B) Genes encoding a Bacillus thuringiensis protein, a derivativethereof or a synthetic polypeptide modeled thereon. See, for example,Geiser, et al., (1986) Gene 48:109, who disclose the cloning andnucleotide sequence of a Bt delta-endotoxin gene. Moreover, DNAmolecules encoding delta-endotoxin genes can be purchased from AmericanType Culture Collection (Rockville, Md.), for example, under ATCC®Accession Numbers 40098, 67136, 31995 and 31998. Other non-limitingexamples of Bacillus thuringiensis transgenes being geneticallyengineered are given in the following patents and patent applicationsand hereby are incorporated by reference for this purpose: U.S. Pat.Nos. 5,188,960; 5,689,052; 5,880,275; 5,986,177; 6,023,013, 6,060,594,6,063,597, 6,077,824, 6,620,988, 6,642,030, 6,713,259, 6,893,826,7,105,332; 7,179,965, 7,208,474; 7,227,056, 7,288,643, 7,323,556,7,329,736, 7,449,552, 7,468,278, 7,510,878, 7,521,235, 7,544,862,7,605,304, 7,696,412, 7,629,504, 7,705,216, 7,772,465, 7,790,846,7,858,849 and WO 1991/14778; WO 1999/31248; WO 2001/12731; WO 1999/24581and WO 1997/40162.

Genes encoding pesticidal proteins may also be stacked including but arenot limited to: insecticidal proteins from Pseudomonas sp. such asPSEEN3174 (Monalysin, (2011) PLoS Pathogens, 7:1-13), from Pseudomonasprotegens strain CHA0 and Pf-5 (previously fluorescens) (Pechy-Tarr,(2008) Environmental Microbiology 10:2368-2386: GenBank Accession No.EU400157); from Pseudomonas Taiwanensis (Liu, et al., (2010) J. Agric.Food Chem. 58:12343-12349) and from Pseudomonas pseudoalcligenes (Zhang,et al., (2009) Annals of Microbiology 59:45-50 and Li, et al., (2007)Plant Cell Tiss. Organ Cult. 89:159-168); insecticidal proteins fromPhotorhabdus sp. and Xenorhabdus sp. (Hinchliffe, et al., (2010) TheOpen Toxinology Journal 3:101-118 and Morgan, et al., (2001) Applied andEnvir. Micro. 67:2062-2069), U.S. Pat. Nos. 6,048,838, and 6,379,946; aPIP-1 polypeptide of U.S. Ser. No. 13/792,861; an AfIP-1A and/or AfIP-1Bpolypeptides of U.S. Ser. No. 13/800,233; a PHI-4 polypeptide of U.S.Ser. No. 13/839,702; PIP-47 polypeptides of U.S. Ser. No. 61/866,747;the insecticidal proteins of U.S. Ser. Nos. 61/863,761 and 61/863763;and δ-endotoxins including, but not limited to, the Cry1, Cry2, Cry3,Cry4, Cry5, Cry6, Cry7, Cry8, Cry9, Cry10, Cry11, Cry12, Cry13, Cry14,Cry15, Cry16, Cry17, Cry18, Cry19, Cry20, Cry21, Cry22, Cry23, Cry24,Cry25, Cry26, Cry27, Cry 28, Cry 29, Cry 30, Cry31, Cry32, Cry33, Cry34,Cry35, Cry36, Cry37, Cry38, Cry39, Cry40, Cry41, Cry42, Cry43, Cry44,Cry45, Cry 46, Cry47, Cry49, Cry 51, Cry52, Cry 53, Cry 54, Cry55,Cry56, Cry57, Cry58, Cry59. Cry60, Cry61, Cry62, Cry63, Cry64, Cry65,Cry66, Cry67, Cry68, Cry69, Cry70 and Cry71 classes of δ-endotoxin genesand the B. thuringiensis cytolytic Cyt1 and Cyt2 genes. Members of theseclasses of B. thuringiensis insecticidal proteins include, but are notlimited to Cry1Aa1 (Accession # AAA22353); Cry1Aa2 (Accession #Accession # AAA22552); Cry1Aa3 (Accession # BAA00257); Cry1Aa4(Accession # CAA31886); Cry1Aa5 (Accession # BAA04468); Cry1Aa6(Accession # AAA86265); Cry1Aa7 (Accession # AAD46139); Cry1Aa8(Accession #I26149); Cry1Aa9 (Accession # BAA77213); Cry1Aa10 (Accession# AAD55382); Cry1Aa11 (Accession # CAA70856); Cry1Aa12 (Accession #AAP80146); Cry1Aa13 (Accession # AAM44305); Cry1Aa14 (Accession #AAP40639); Cry1Aa15 (Accession # AAY66993); Cry1Aa16 (Accession #HQ439776); Cry1Aa17 (Accession # HQ439788); Cry1Aa18 (Accession #HQ439790); Cry1Aa19 (Accession # HQ685121); Cry1Aa20 (Accession #JF340156); Cry1Aa21 (Accession # JN651496); Cry1Aa22 (Accession #KC158223); Cry1Ab1 (Accession # AAA22330); Cry1Ab2 (Accession #AAA22613); Cry1Ab3 (Accession # AAA22561); Cry1Ab4 (Accession #BAA00071); Cry1Ab5 (Accession # CAA28405); Cry1Ab6 (Accession #AAA22420); Cry1Ab7 (Accession # CAA31620); Cry1Ab8 (Accession #AAA22551); Cry1Ab9 (Accession # CAA38701); Cry1Ab10 (Accession #A29125); Cry1Ab11 (Accession #I12419); Cry1Ab12 (Accession # AAC64003);Cry1Ab13 (Accession # AAN76494); Cry1Ab14 (Accession # AAG16877);Cry1Ab15 (Accession # AAO13302); Cry1Ab16 (Accession # AAK55546);Cry1Ab17 (Accession # AAT46415); Cry1Ab18 (Accession # AAQ88259);Cry1Ab19 (Accession # AAW31761); Cry1Ab20 (Accession # ABB72460);Cry1Ab21 (Accession # ABS18384); Cry1Ab22 (Accession # ABW87320);Cry1Ab23 (Accession # HQ439777); Cry1Ab24 (Accession # HQ439778);Cry1Ab25 (Accession # HQ685122); Cry1Ab26 (Accession # HQ847729);Cry1Ab27 (Accession # JN135249); Cry1Ab28 (Accession # JN135250);Cry1Ab29 (Accession # JN135251); Cry1Ab30 (Accession # JN135252);Cry1Ab31 (Accession # JN135253); Cry1Ab32 (Accession # JN135254);Cry1Ab33 (Accession # AAS93798); Cry1Ab34 (Accession # KC156668);Cry1Ab-like (Accession # AAK14336); Cry1Ab-like (Accession # AAK14337);Cry1Ab-like (Accession # AAK14338); Cry1Ab-like (Accession # ABG88858);Cry1Ac1 (Accession # AAA22331); Cry1Ac2 (Accession # AAA22338); Cry1Ac3(Accession # CAA38098); Cry1Ac4 (Accession # AAA73077); Cry1Ac5(Accession # AAA22339); Cry1Ac6 (Accession # AAA86266); Cry1Ac7(Accession # AAB46989); Cry1Ac8 (Accession # AAC44841); Cry1Ac9(Accession # AAB49768); Cry1Ac10 (Accession # CAA05505); Cry1Ac11(Accession # CAA10270); Cry1Ac12 (Accession #I12418); Cry1Ac13(Accession # AAD38701); Cry1Ac14 (Accession # AAQ06607); Cry1Ac15(Accession # AAN07788); Cry1Ac16 (Accession # AAU87037); Cry1Ac17(Accession # AAX18704); Cry1Ac18 (Accession # AAY88347); Cry1Ac19(Accession # ABD37053); Cry1Ac20 (Accession # ABB89046); Cry1Ac21(Accession # AAY66992); Cry1Ac22 (Accession # ABZ01836); Cry1Ac23(Accession # CAQ30431); Cry1Ac24 (Accession # ABL01535); Cry1Ac25(Accession # FJ513324); Cry1Ac26 (Accession # FJ617446); Cry1Ac27(Accession # FJ617447); Cry1Ac28 (Accession # ACM90319); Cry1Ac29(Accession # DQ438941); Cry1Ac30 (Accession # GQ227507); Cry1Ac31(Accession # GU446674); Cry1Ac32 (Accession # HM061081); Cry1Ac33(Accession # GQ866913); Cry1Ac34 (Accession # HQ230364); Cry1Ac35(Accession # JF340157); Cry1Ac36 (Accession # JN387137); Cry1Ac37(Accession # JQ317685); Cry1Ad1 (Accession # AAA22340); Cry1Ad2(Accession # CAA01880); Cry1Ae1 (Accession # AAA22410); Cry1Af1(Accession # AAB82749); Cry1Ag1 (Accession # AAD46137); Cry1Ah1(Accession # AAQ14326); Cry1Ah2 (Accession # ABB76664); Cry1Ah3(Accession # HQ439779); Cry1Ai1 (Accession # AAO39719); Cry1Ai2(Accession # HQ439780); Cry1A-like (Accession # AAK14339); Cry1Ba1(Accession # CAA29898); Cry1Ba2 (Accession # CAA65003); Cry1Ba3(Accession # AAK63251); Cry1Ba4 (Accession # AAK51084); Cry1Ba5(Accession # AB020894); Cry1Ba6 (Accession # ABL60921); Cry1Ba7(Accession # HQ439781); Cry1Bb1 (Accession # AAA22344); Cry1Bb2(Accession # HQ439782); Cry1Bc1 (Accession # CAA86568); Cry1Bd1(Accession # AAD10292); Cry1Bd2 (Accession # AAM93496); Cry1Be1(Accession # AAC32850); Cry1Be2 (Accession # AAQ52387); Cry1Be3(Accession # ACV96720); Cry1Be4 (Accession # HM070026); Cry1Bf1(Accession # CAC50778); Cry1Bf2 (Accession # AAQ52380); Cry1Bg1(Accession # AAO39720); Cry1Bh1 (Accession # HQ589331); Cry1Bi1(Accession # KC156700); Cry1Ca1 (Accession # CAA30396); Cry1Ca2(Accession # CAA31951); Cry1Ca3 (Accession # AAA22343); Cry1Ca4(Accession # CAA01886); Cry1Ca5 (Accession # CAA65457); Cry1Ca6[1](Accession # AAF37224); Cry1Ca7 (Accession # AAG50438); Cry1Ca8(Accession # AAM00264); Cry1Ca9 (Accession # AAL79362); Cry1Ca10(Accession # AAN16462); Cry1Ca11 (Accession # AAX53094); Cry1Ca12(Accession # HM070027); Cry1Ca13 (Accession # HQ412621); Cry1Ca14(Accession # JN651493); Cry1Cb1 (Accession # M97880); Cry1Cb2 (Accession# AAG35409); Cry1Cb3 (Accession # ACD50894); Cry1Cb-like (Accession #AAX63901); Cry1Da1 (Accession # CAA38099); Cry1Da2 (Accession #I76415);Cry1Da3 (Accession # HQ439784); Cry1Db1 (Accession # CAA80234); Cry1 Db2(Accession # AAK48937); Cry1 Dc1 (Accession # ABK35074); Cry1Ea1(Accession # CAA37933); Cry1Ea2 (Accession # CAA39609); Cry1Ea3(Accession # AAA22345); Cry1Ea4 (Accession # AAD04732); Cry1Ea5(Accession # A15535); Cry1Ea6 (Accession # AAL50330); Cry1Ea7 (Accession# AAW72936); Cry1Ea8 (Accession # ABX11258); Cry1Ea9 (Accession #HQ439785); Cry1Ea10 (Accession # ADR00398); Cry1Ea11 (Accession #JQ652456); Cry1Eb1 (Accession # AAA22346); Cry1Fa1 (Accession #AAA22348); Cry1Fa2 (Accession # AAA22347); Cry1Fa3 (Accession #HM070028); Cry1Fa4 (Accession # HM439638); Cry1Fb1 (Accession #CAA80235); Cry1 Fb2 (Accession # BAA25298); Cry1 Fb3 (Accession #AAF21767); Cry1Fb4 (Accession # AAC10641); Cry1Fb5 (Accession #AAO13295); Cry1Fb6 (Accession # ACD50892); Cry1Fb7 (Accession #ACD50893); Cry1Ga1 (Accession # CAA80233); Cry1Ga2 (Accession #CAA70506); Cry1Gb1 (Accession # AAD10291); Cry1Gb2 (Accession #AAO13756); Cry1Gc1 (Accession # AAQ52381); Cry1 Ha1 (Accession #CAA80236); Cry1 Hb1 (Accession # AAA79694); Cry1 Hb2 (Accession #HQ439786); Cry1H-like (Accession # AAF01213); Cry1Ia1 (Accession #CAA44633); Cry1Ia2 (Accession # AAA22354); Cry1Ia3 (Accession #AAC36999); Cry1Ia4 (Accession # AAB00958); Cry1Ia5 (Accession #CAA70124); Cry1Ia6 (Accession # AAC26910); Cry1Ia7 (Accession #AAM73516); Cry1Ia8 (Accession # AAK66742); Cry1Ia9 (Accession #AAQ08616); Cry1Ia10 (Accession # AAP86782); Cry1Ia11 (Accession #CAC85964); Cry1Ia12 (Accession # AAV53390); Cry1Ia13 (Accession #ABF83202); Cry1Ia14 (Accession # ACG63871); Cry1Ia15 (Accession #FJ617445); Cry1Ia16 (Accession # FJ617448); Cry1Ia17 (Accession #GU989199); Cry1Ia18 (Accession # ADK23801); Cry1Ia19 (Accession #HQ439787); Cry1Ia20 (Accession # JQ228426); Cry1Ia21 (Accession #JQ228424); Cry1Ia22 (Accession # JQ228427); Cry1Ia23 (Accession #JQ228428); Cry1Ia24 (Accession # JQ228429); Cry1Ia25 (Accession #JQ228430); Cry1Ia26 (Accession # JQ228431); Cry1Ia27 (Accession #JQ228432); Cry1Ia28 (Accession # JQ228433); Cry1Ia29 (Accession #JQ228434); Cry1Ia30 (Accession # JQ317686); Cry1Ia31 (Accession #JX944038); Cry1Ia32 (Accession # JX944039); Cry1Ia33 (Accession #JX944040); Cry1Ib1 (Accession # AAA82114); Cry1Ib12 (Accession #ABW88019); Cry1Ib13 (Accession # ACD75515); Cry1Ib14 (Accession #HM051227); Cry1Ib15 (Accession # HM070028); Cry1Ib16 (Accession #ADK38579); Cry1Ib17 (Accession # JN571740); Cry1Ib8 (Accession #JN675714); Cry1Ib9 (Accession # JN675715); Cry1Ib10 (Accession #JN675716); Cry1Ib11 (Accession # JQ228423); Cry1Ic1 (Accession #AAC62933); Cry1Ic2 (Accession # AAE71691); Cry1 dl (Accession #AAD44366); Cry1Id2 (Accession # JQ228422); Cry1Ie1 (Accession #AAG43526); Cry1Ie2 (Accession # HM439636); Cry1Ie3 (Accession #KC156647); Cry1Ie4 (Accession # KC156681); Cry1If1 (Accession #AAQ52382); Cry1Ig1 (Accession # KC156701); Cry1I-like (Accession #AAC31094); Cry1I-like (Accession # ABG88859); Cry1Ja1 (Accession #AAA22341); Cry1Ja2 (Accession # HM070030); Cry1Ja3 (Accession #JQ228425); Cry1Jb1 (Accession # AAA98959); Cry1Jc1 (Accession #AAC31092); Cry1Jc2 (Accession # AAQ52372); Cry1Jd1 (Accession #CAC50779); Cry1 Ka1 (Accession # AAB00376); Cry1 Ka2 (Accession #HQ439783); Cry1La1 (Accession # AAS60191); Cry1La2 (Accession #HM070031); Cry1Ma1 (Accession # FJ884067); Cry1Ma2 (Accession #KC156659); Cry1Na1 (Accession # KC156648); Cry1Nb1 (Accession #KC156678); Cry1-like (Accession # AAC31091); Cry2Aa1 (Accession #AAA22335); Cry2Aa2 (Accession # AAA83516); Cry2Aa3 (Accession # D86064);Cry2Aa4 (Accession # AAC04867); Cry2Aa5 (Accession # CAA10671); Cry2Aa6(Accession # CAA10672); Cry2Aa7 (Accession # CAA10670); Cry2Aa8(Accession # AAO13734); Cry2Aa9 (Accession # AAO13750); Cry2Aa10(Accession # AAQ04263); Cry2Aa11 (Accession # AAQ52384); Cry2Aa12(Accession # AB183671); Cry2Aa13 (Accession # ABL01536); Cry2Aa14(Accession # ACF04939); Cry2Aa15 (Accession # JN426947); Cry2Ab1(Accession # AAA22342); Cry2Ab2 (Accession # CAA39075); Cry2Ab3(Accession # AAG36762); Cry2Ab4 (Accession # AAO13296); Cry2Ab5(Accession # AAQ04609); Cry2Ab6 (Accession # AAP59457); Cry2Ab7(Accession # AAZ66347); Cry2Ab8 (Accession # ABC95996); Cry2Ab9(Accession # ABC74968); Cry2Ab10 (Accession # EF157306); Cry2Ab11(Accession # CAM84575); Cry2Ab12 (Accession # ABM21764); Cry2Ab13(Accession # ACG76120); Cry2Ab14 (Accession # ACG76121); Cry2Ab15(Accession # HM037126); Cry2Ab16 (Accession # GQ866914); Cry2Ab17(Accession # HQ439789); Cry2Ab18 (Accession # JN135255); Cry2Ab19(Accession # JN135256); Cry2Ab20 (Accession # JN135257); Cry2Ab21(Accession # JN135258); Cry2Ab22 (Accession # JN135259); Cry2Ab23(Accession # JN135260); Cry2Ab24 (Accession # JN135261); Cry2Ab25(Accession # JN415485); Cry2Ab26 (Accession # JN426946); Cry2Ab27(Accession # JN415764); Cry2Ab28 (Accession # JN651494); Cry2Ac1(Accession # CAA40536); Cry2Ac2 (Accession # AAG35410); Cry2Ac3(Accession # AAQ52385); Cry2Ac4 (Accession # ABC95997); Cry2Ac5(Accession # ABC74969); Cry2Ac6 (Accession # ABC74793); Cry2Ac7(Accession # CAL18690); Cry2Ac8 (Accession # CAM09325); Cry2Ac9(Accession # CAM09326); Cry2Ac10 (Accession # ABN15104); Cry2Ac11(Accession # CAM83895); Cry2Ac12 (Accession # CAM83896); Cry2Ad1(Accession # AAF09583); Cry2Ad2 (Accession # ABC86927); Cry2Ad3(Accession # CAK29504); Cry2Ad4 (Accession # CAM32331); Cry2Ad5(Accession # CAO78739); Cry2Ae1 (Accession # AAQ52362); Cry2Af1(Accession # AB030519); Cry2Af2 (Accession # GQ866915); Cry2Ag1(Accession # ACH91610); Cry2Ah1 (Accession # EU939453); Cry2Ah2(Accession # ACL80665); Cry2Ah3 (Accession # GU073380); Cry2Ah4(Accession # KC156702); Cry2Ai1 (Accession # FJ788388); Cry2Aj(Accession #); Cry2Ak1 (Accession # KC156660); Cry2Ba1 (Accession #KC156658); Cry3Aa1 (Accession # AAA22336); Cry3Aa2 (Accession #AAA22541); Cry3Aa3 (Accession # CAA68482); Cry3Aa4 (Accession #AAA22542); Cry3Aa5 (Accession # AAA50255); Cry3Aa6 (Accession #AAC43266); Cry3Aa7 (Accession # CAB41411); Cry3Aa8 (Accession #AAS79487); Cry3Aa9 (Accession # AAW05659); Cry3Aa10 (Accession #AAU29411); Cry3Aa11 (Accession # AAW82872); Cry3Aa12 (Accession #ABY49136); Cry3Ba1 (Accession # CAA34983); Cry3Ba2 (Accession #CAA00645); Cry3Ba3 (Accession # JQ397327); Cry3Bb1 (Accession #AAA22334); Cry3Bb2 (Accession # AAA74198); Cry3Bb3 (Accession #I15475);Cry3Ca1 (Accession # CAA42469); Cry4Aa1 (Accession # CAA68485); Cry4Aa2(Accession # BAA00179); Cry4Aa3 (Accession # CAD30148); Cry4Aa4(Accession # AFB18317); Cry4A-like (Accession # AAY96321); Cry4Ba1(Accession # CAA30312); Cry4Ba2 (Accession # CAA30114); Cry4Ba3(Accession # AAA22337); Cry4Ba4 (Accession # BAA00178); Cry4Ba5(Accession # CAD30095); Cry4Ba-like (Accession # ABC47686); Cry4Ca1(Accession # EU646202); Cry4Cb1 (Accession # FJ403208); Cry4Cb2(Accession # FJ597622); Cry4Cc1 (Accession # FJ403207); Cry5Aa1(Accession # AAA67694); Cry5Ab1 (Accession # AAA67693); Cry5Ac1(Accession #I34543); Cry5Ad1 (Accession # ABQ82087); Cry5Ba1 (Accession# AAA68598); Cry5Ba2 (Accession # ABW88931); Cry5Ba3 (Accession #AFJ04417); Cry5Ca1 (Accession # HM461869); Cry5Ca2 (Accession #ZP_04123426); Cry5Da1 (Accession # HM461870); Cry5Da2 (Accession #ZP_04123980); Cry5Ea1 (Accession # HM485580); Cry5Ea2 (Accession #ZP_04124038); Cry6Aa1 (Accession # AAA22357); Cry6Aa2 (Accession #AAM46849); Cry6Aa3 (Accession # ABH03377); Cry6Ba1 (Accession #AAA22358); Cry7Aa1 (Accession # AAA22351); Cry7Ab1 (Accession #AAA21120); Cry7Ab2 (Accession # AAA21121); Cry7Ab3 (Accession #ABX24522); Cry7Ab4 (Accession # EU380678); Cry7Ab5 (Accession #ABX79555); Cry7Ab6 (Accession # ACI44005); Cry7Ab7 (Accession #ADB89216); Cry7Ab8 (Accession # GU145299); Cry7Ab9 (Accession #ADD92572); Cry7Ba1 (Accession # ABB70817); Cry7Bb1 (Accession #KC156653); Cry7Ca1 (Accession # ABR67863); Cry7Cb1 (Accession #KC156698); Cry7Da1 (Accession # ACQ99547); Cry7Da2 (Accession #HM572236); Cry7Da3 (Accession # KC156679); Cry7Ea1 (Accession #HM035086); Cry7Ea2 (Accession # HM132124); Cry7Ea3 (Accession #EEM19403); Cry7Fa1 (Accession # HM035088); Cry7Fa2 (Accession #EEM19090); Cry7Fb1 (Accession # HM572235); Cry7Fb2 (Accession #KC156682); Cry7Ga1 (Accession # HM572237); Cry7Ga2 (Accession #KC156669); Cry7Gb1 (Accession # KC156650); Cry7Gc1 (Accession #KC156654); Cry7Gd1 (Accession # KC156697); Cry7Ha1 (Accession #KC156651); Cry7Ia1 (Accession # KC156665); Cry7Ja1 (Accession #KC156671); Cry7Ka1 (Accession # KC156680); Cry7Kb1 (Accession #BAM99306); Cry7La1 (Accession # BAM99307); Cry8Aa1 (Accession #AAA21117); Cry8Ab1 (Accession # EU044830); Cry8Ac1 (Accession #KC156662); Cry8Ad1 (Accession # KC156684); Cry8Ba1 (Accession #AAA21118); Cry8Bb1 (Accession # CAD57542); Cry8Bc1 (Accession #CAD57543); Cry8Ca1 (Accession # AAA21119); Cry8Ca2 (Accession #AAR98783); Cry8Ca3 (Accession # EU625349); Cry8Ca4 (Accession #ADB54826); Cry8Da1 (Accession # BAC07226); Cry8Da2 (Accession #BD133574); Cry8Da3 (Accession # BD133575); Cry8Db1 (Accession #BAF93483); Cry8Ea1 (Accession # AAQ73470); Cry8Ea2 (Accession #EU047597); Cry8Ea3 (Accession # KC855216); Cry8Fa1 (Accession #AAT48690); Cry8Fa2 (Accession # HQ174208); Cry8Fa3 (Accession #AFH78109); Cry8Ga1 (Accession # AAT46073); Cry8Ga2 (Accession #ABC42043); Cry8Ga3 (Accession # FJ198072); Cry8Ha1 (Accession #AAW81032); Cry8Ia1 (Accession # EU381044); Cry8Ia2 (Accession #GU073381); Cry8Ia3 (Accession # HM044664); Cry8Ia4 (Accession #KC156674); Cry8Ib1 (Accession # GU325772); Cry8Ib2 (Accession #KC156677); Cry8Ja1 (Accession # EU625348); Cry8Ka1 (Accession #FJ422558); Cry8Ka2 (Accession # ACN87262); Cry8Kb1 (Accession #HM123758); Cry8Kb2 (Accession # KC156675); Cry8La1 (Accession #GU325771); Cry8Ma1 (Accession # HM044665); Cry8Ma2 (Accession #EEM86551); Cry8Ma3 (Accession # HM210574); Cry8Na1 (Accession #HM640939); Cry8Pa1 (Accession # HQ388415); Cry8Qa1 (Accession #HQ441166); Cry8Qa2 (Accession # KC152468); Cry8Ra1 (Accession #AFP87548); Cry8Sa1 (Accession # JQ740599); Cry8Ta1 (Accession #KC156673); Cry8-like (Accession # FJ770571); Cry8-like (Accession #ABS53003); Cry9Aa1 (Accession # CAA41122); Cry9Aa2 (Accession #CAA41425); Cry9Aa3 (Accession # GQ249293); Cry9Aa4 (Accession #GQ249294); Cry9Aa5 (Accession # JX174110); Cry9Aa like (Accession #AAQ52376); Cry9Ba1 (Accession # CAA52927); Cry9Ba2 (Accession #GU299522); Cry9Bb1 (Accession # AAV28716); Cry9Ca1 (Accession #CAA85764); Cry9Ca2 (Accession # AAQ52375); Cry9Da1 (Accession #BAA19948); Cry9Da2 (Accession # AAB97923); Cry9Da3 (Accession #GQ249293); Cry9Da4 (Accession # GQ249297); Cry9Db1 (Accession #AAX78439); Cry9Dc1 (Accession # KC156683); Cry9Ea1 (Accession #BAA34908); Cry9Ea2 (Accession # AAO12908); Cry9Ea3 (Accession #ABM21765); Cry9Ea4 (Accession # ACE88267); Cry9Ea5 (Accession #ACF04743); Cry9Ea6 (Accession # ACG63872); Cry9Ea7 (Accession #FJ380927); Cry9Ea8 (Accession # GQ249292); Cry9Ea9 (Accession #JN651495); Cry9Eb1 (Accession # CAC50780); Cry9Eb2 (Accession #GQ249298); Cry9Eb3 (Accession # KC156646); Cry9Ec1 (Accession #AAC63366); Cry9Ed1 (Accession # AAX78440); Cry9Ee1 (Accession #GQ249296); Cry9Ee2 (Accession # KC156664); Cry9Fa1 (Accession #KC156692); Cry9Ga1 (Accession # KC156699); Cry9-like (Accession #AAC63366); Cry10Aa1 (Accession # AAA22614); Cry10Aa2 (Accession #E00614); Cry10Aa3 (Accession # CAD30098); Cry10Aa4 (Accession #AFB18318); Cry10A-like (Accession # DQ167578); Cry11Aa1 (Accession #AAA22352); Cry11Aa2 (Accession # AAA22611); Cry11Aa3 (Accession #CAD30081); Cry11Aa4 (Accession # AFB18319); Cry11Aa-like (Accession #DQ166531); Cry11Ba1 (Accession # CAA60504); Cry11Bb1 (Accession #AAC97162); Cry11Bb2 (Accession # HM068615); Cry12Aa1 (Accession #AAA22355); Cry13Aa1 (Accession # AAA22356); Cry14Aa1 (Accession #AAA21516); Cry14Ab1 (Accession # KC156652); Cry15Aa1 (Accession #AAA22333); Cry16Aa1 (Accession # CAA63860); Cry17Aa1 (Accession #CAA67841); Cry18Aa1 (Accession # CAA67506); Cry18Ba1 (Accession #AAF89667); Cry18Ca1 (Accession # AAF89668); Cry19Aa1 (Accession #CAA68875); Cry19Ba1 (Accession # BAA32397); Cry19Ca1 (Accession #AFM37572); Cry20Aa1 (Accession # AAB93476); Cry20Ba1 (Accession #ACS93601); Cry20Ba2 (Accession # KC156694); Cry20-like (Accession #GQ144333); Cry21Aa1 (Accession #I32932); Cry21Aa2 (Accession #I66477);Cry21Ba1 (Accession # BAC06484); Cry21Ca1 (Accession # JF521577);Cry21Ca2 (Accession # KC156687); Cry21Da1 (Accession # JF521578);Cry22Aa1 (Accession #I34547); Cry22Aa2 (Accession # CAD43579); Cry22Aa3(Accession # ACD93211); Cry22Ab1 (Accession # AAK50456); Cry22Ab2(Accession # CAD43577); Cry22Ba1 (Accession # CAD43578); Cry22Bb1(Accession # KC156672); Cry23Aa1 (Accession # AAF76375); Cry24Aa1(Accession # AAC61891); Cry24Ba1 (Accession # BAD32657); Cry24Ca1(Accession # CAJ43600); Cry25Aa1 (Accession # AAC61892); Cry26Aa1(Accession # AAD25075); Cry27Aa1 (Accession # BAA82796); Cry28Aa1(Accession # AAD24189); Cry28Aa2 (Accession # AAG00235); Cry29Aa1(Accession # CAC80985); Cry30Aa1 (Accession # CAC80986); Cry30Ba1(Accession # BAD00052); Cry30Ca1 (Accession # BAD67157); Cry30Ca2(Accession # ACU24781); Cry30Da1 (Accession # EF095955); Cry30Db1(Accession # BAE80088); Cry30Ea1 (Accession # ACC95445); Cry30Ea2(Accession # FJ499389); Cry30Fa1 (Accession # ACI22625); Cry30Ga1(Accession # ACG60020); Cry30Ga2 (Accession # HQ638217); Cry31Aa1(Accession # BAB11757); Cry31Aa2 (Accession # AAL87458); Cry31Aa3(Accession # BAE79808); Cry31Aa4 (Accession # BAF32571); Cry31Aa5(Accession # BAF32572); Cry31Aa6 (Accession # BA144026); Cry31Ab1(Accession # BAE79809); Cry31Ab2 (Accession # BAF32570); Cry31Ac1(Accession # BAF34368); Cry31Ac2 (Accession # AB731600); Cry31Ad1(Accession # BA144022); Cry32Aa1 (Accession # AAG36711); Cry32Aa2(Accession # GU063849); Cry32Ab1 (Accession # GU063850); Cry32Ba1(Accession # BAB78601); Cry32Ca1 (Accession # BAB78602); Cry32Cb1(Accession # KC156708); Cry32Da1 (Accession # BAB78603); Cry32Ea1(Accession # GU324274); Cry32Ea2 (Accession # KC156686); Cry32Eb1(Accession # KC156663); Cry32Fa1 (Accession # KC156656); Cry32Ga1(Accession # KC156657); Cry32Ha1 (Accession # KC156661); Cry32Hb1(Accession # KC156666); Cry32Ia1 (Accession # KC156667); Cry32Ja1(Accession # KC156685); Cry32Ka1 (Accession # KC156688); Cry32La1(Accession # KC156689); Cry32Ma1 (Accession # KC156690); Cry32Mb1(Accession # KC156704); Cry32Na1 (Accession # KC156691); Cry32Oa1(Accession # KC156703); Cry32Pa1 (Accession # KC156705); Cry32Qa1(Accession # KC156706); Cry32Ra1 (Accession # KC156707); Cry32Sa1(Accession # KC156709); Cry32Ta1 (Accession # KC156710); Cry32Ua1(Accession # KC156655); Cry33Aa1 (Accession # AAL26871); Cry34Aa1(Accession # AAG50341); Cry34Aa2 (Accession # AAK64560); Cry34Aa3(Accession # AAT29032); Cry34Aa4 (Accession # AAT29030); Cry34Ab1(Accession # AAG41671); Cry34Ac1 (Accession # AAG50118); Cry34Ac2(Accession # AAK64562); Cry34Ac3 (Accession # AAT29029); Cry34Ba1(Accession # AAK64565); Cry34Ba2 (Accession # AAT29033); Cry34Ba3(Accession # AAT29031); Cry35Aa1 (Accession # AAG50342); Cry35Aa2(Accession # AAK64561); Cry35Aa3 (Accession # AAT29028); Cry35Aa4(Accession # AAT29025); Cry35Ab1 (Accession # AAG41672); Cry35Ab2(Accession # AAK64563); Cry35Ab3 (Accession # AY536891); Cry35Ac1(Accession # AAG50117); Cry35Ba1 (Accession # AAK64566); Cry35Ba2(Accession # AAT29027); Cry35Ba3 (Accession # AAT29026); Cry36Aa1(Accession # AAK64558); Cry37Aa1 (Accession # AAF76376); Cry38Aa1(Accession # AAK64559); Cry39Aa1 (Accession # BAB72016); Cry40Aa1(Accession # BAB72018); Cry40Ba1 (Accession # BAC77648); Cry40Ca1(Accession # EU381045); Cry40Da1 (Accession # ACF15199); Cry41Aa1(Accession # BAD35157); Cry41Ab1 (Accession # BAD35163); Cry41Ba1(Accession # HM461871); Cry41Ba2 (Accession # ZP_04099652); Cry42Aa1(Accession # BAD35166); Cry43Aa1 (Accession # BAD15301); Cry43Aa2(Accession # BAD95474); Cry43Ba1 (Accession # BAD15303); Cry43Ca1(Accession # KC156676); Cry43Cb1 (Accession # KC156695); Cry43Cc1(Accession # KC156696); Cry43-like (Accession # BAD15305); Cry44Aa(Accession # BAD08532); Cry45Aa (Accession # BAD22577); Cry46Aa(Accession # BAC79010); Cry46Aa2 (Accession # BAG68906); Cry46Ab(Accession # BAD35170); Cry47Aa (Accession # AAY24695); Cry48Aa(Accession # CAJ18351); Cry48Aa2 (Accession # CAJ86545); Cry48Aa3(Accession # CAJ86546); Cry48Ab (Accession # CAJ86548); Cry48Ab2(Accession # CAJ86549); Cry49Aa (Accession # CAH56541); Cry49Aa2(Accession # CAJ86541); Cry49Aa3 (Accession # CAJ86543); Cry49Aa4(Accession # CAJ86544); Cry49Ab1 (Accession # CAJ86542); Cry50Aa1(Accession # BAE86999); Cry50Ba1 (Accession # GU446675); Cry50Ba2(Accession # GU446676); Cry51Aa1 (Accession # AB114444); Cry51Aa2(Accession # GU570697); Cry52Aa1 (Accession # EF613489); Cry52Ba1(Accession # FJ361760); Cry53Aa1 (Accession # EF633476); Cry53Ab1(Accession # FJ361759); Cry54Aa1 (Accession # ACA52194); Cry54Aa2(Accession # GQ140349); Cry54Ba1 (Accession # GU446677); Cry55Aa1(Accession # ABW88932); Cry54Ab1 (Accession # JQ916908); Cry55Aa2(Accession # AAE33526); Cry56Aa1 (Accession # ACU57499); Cry56Aa2(Accession # GQ483512); Cry56Aa3 (Accession # JX025567); Cry57Aa1(Accession # ANC87261); Cry58Aa1 (Accession # ANC87260); Cry59Ba1(Accession # JN790647); Cry59Aa1 (Accession # ACR43758); Cry60Aa1(Accession # ACU24782); Cry60Aa2 (Accession # EAO57254); Cry60Aa3(Accession # EEM99278); Cry60Ba1 (Accession # GU810818); Cry60Ba2(Accession # EAO57253); Cry60Ba3 (Accession # EEM99279); Cry61Aa1(Accession # HM035087); Cry61Aa2 (Accession # HM132125); Cry61Aa3(Accession # EEM19308); Cry62Aa1 (Accession # HM054509); Cry63Aa1(Accession # BA144028); Cry64Aa1 (Accession # BAJ05397); Cry65Aa1(Accession # HM461868); Cry65Aa2 (Accession # ZP_04123838); Cry66Aa1(Accession # HM485581); Cry66Aa2 (Accession # ZP_04099945); Cry67Aa1(Accession # HM485582); Cry67Aa2 (Accession # ZP_04148882); Cry68Aa1(Accession # HQ113114); Cry69Aa1 (Accession # HQ401006); Cry69Aa2(Accession # JQ821388); Cry69Ab1 (Accession # JN209957); Cry70Aa1(Accession # JN646781); Cry70Ba1 (Accession # ADO51070); Cry70Bb1(Accession # EEL67276); Cry71Aa1 (Accession # JX025568); Cry72Aa1(Accession # JX025569).

Examples of δ-endotoxins also include but are not limited to Cry1Aproteins of U.S. Pat. Nos. 5,880,275, 7,858,849 8,530,411, 8,575,433,and 8,686,233; a DIG-3 or DIG-11 toxin (N-terminal deletion of α-helix 1and/or α-helix 2 variants of cry proteins such as Cry1A, Cry3A) of U.S.Pat. Nos. 8,304,604, 8,304,605 and 8,476,226; Cry1B of U.S. patentapplication Ser. No. 10/525,318; Cry1C of U.S. Pat. No. 6,033,874; Cry1Fof U.S. Pat. Nos. 5,188,960 and 6,218,188; Cry1A/F chimeras of U.S. Pat.Nos. 7,070,982; 6,962,705 and 6,713,063); a Cry2 protein such as Cry2Abprotein of U.S. Pat. No. 7,064,249); a Cry3A protein including but notlimited to an engineered hybrid insecticidal protein (eHIP) created byfusing unique combinations of variable regions and conserved blocks ofat least two different Cry proteins (US Patent Application PublicationNumber 2010/0017914); a Cry4 protein; a Cry5 protein; a Cry6 protein;Cry8 proteins of U.S. Pat. Nos. 7,329,736, 7,449,552, 7,803,943,7,476,781, 7,105,332, 7,378,499 and 7,462,760; a Cry9 protein such assuch as members of the Cry9A, Cry9B, Cry9C, Cry9D, Cry9E and Cry9Ffamilies, including but not limited to the Cry9D protein of U.S. Pat.No. 8,802,933 and the Cry9B protein of U.S. Pat. No. 8,802,934; a Cry15protein of Naimov, et al., (2008) Applied and EnvironmentalMicrobiology, 74:7145-7151; a Cry22, a Cry34Ab1 protein of U.S. Pat.Nos. 6,127,180, 6,624,145 and 6,340,593; a CryET33 and cryET34 proteinof U.S. Pat. Nos. 6,248,535, 6,326,351, 6,399,330, 6,949,626, 7,385,107and 7,504,229; a CryET33 and CryET34 homologs of US Patent PublicationNumber 2006/0191034, 2012/0278954, and PCT Publication Number WO2012/139004; a Cry35Ab1 protein of U.S. Pat. Nos. 6,083,499, 6,548,291and 6,340,593; a Cry46 protein, a Cry 51 protein, a Cry binary toxin; aTIC901 or related toxin; TIC807 of US Patent Application PublicationNumber 2008/0295207; ET29, ET37, TIC809, TIC810, TIC812, TIC127, TIC128of PCT US 2006/033867; TIC853 toxins of U.S. Pat. No. 8,513,494,AXMI-027, AXMI-036, and AXMI-038 of U.S. Pat. No. 8,236,757; AXMI-031,AXMI-039, AXMI-040, AXMI-049 of U.S. Pat. No. 7,923,602; AXMI-018,AXMI-020 and AXMI-021 of WO 2006/083891; AXMI-010 of WO 2005/038032;AXMI-003 of WO 2005/021585; AXMI-008 of US Patent ApplicationPublication Number 2004/0250311; AXMI-006 of US Patent ApplicationPublication Number 2004/0216186; AXMI-007 of US Patent ApplicationPublication Number 2004/0210965; AXMI-009 of US Patent ApplicationNumber 2004/0210964; AXMI-014 of US Patent Application PublicationNumber 2004/0197917; AXMI-004 of US Patent Application PublicationNumber 2004/0197916; AXMI-028 and AXMI-029 of WO 2006/119457; AXMI-007,AXMI-008, AXMI-0080rf2, AXMI-009, AXMI-014 and AXMI-004 of WO2004/074462; AXMI-150 of U.S. Pat. No. 8,084,416; AXMI-205 of US PatentApplication Publication Number 2011/0023184; AXMI-011, AXMI-012,AXMI-013, AXMI-015, AXMI-019, AXMI-044, AXMI-037, AXMI-043, AXMI-033,AXMI-034, AXMI-022, AXMI-023, AXMI-041, AXMI-063 and AXMI-064 of USPatent Application Publication Number 2011/0263488; AXMI-R1 and relatedproteins of US Patent Application Publication Number 2010/0197592;AXMI221Z, AXMI222z, AXMI223z, AXMI224z and AXMI225z of WO 2011/103248;AXMI218, AXMI219, AXMI220, AXMI226, AXMI227, AXMI228, AXMI229, AXMI230and AXMI231 of WO 2011/103247 and U.S. Pat. No. 8,759,619; AXMI-115,AXMI-113, AXMI-005, AXMI-163 and AXMI-184 of U.S. Pat. No. 8,334,431;AXMI-001, AXMI-002, AXMI-030, AXMI-035 and AXMI-045 of US PatentApplication Publication Number 2010/0298211; AXMI-066 and AXMI-076 of USPatent Application Publication Number 2009/0144852; AXMI128, AXMI130,AXMI131, AXMI133, AXMI140, AXMI141, AXMI142, AXMI143, AXMI144, AXMI146,AXMI148, AXMI149, AXMI152, AXMI153, AXMI154, AXMI155, AXMI156, AXMI157,AXMI158, AXMI162, AXMI165, AXMI166, AXMI167, AXMI168, AXMI169, AXMI170,AXMI171, AXMI172, AXMI173, AXMI174, AXMI175, AXMI176, AXMI177, AXMI178,AXMI179, AXMI180, AXMI181, AXMI182, AXMI185, AXMI186, AXMI187, AXMI188,AXMI189 of U.S. Pat. No. 8,318,900; AXMI079, AXMI080, AXMI081, AXMI082,AXMI091, AXMI092, AXMI096, AXMI097, AXMI098, AXMI099, AXMI100, AXMI101,AXMI102, AXMI103, AXMI104, AXMI107, AXMI108, AXMI109, AXMI110, AXMI111,AXMI112, AXMI114, AXMI116, AXMI117, AXMI118, AXMI119, AXMI120, AXMI121,AXMI122, AXMI123, AXMI124, AXMI1257, AXMI1268, AXMI127, AXMI129,AXMI164, AXMI151, AXMI161, AXMI183, AXMI132, AXMI138, AXMI137 of USPatent Application Publication Number 2010/0005543, AXMI270 of US PatentApplication Publication US20140223598, AXMI279 of US Patent ApplicationPublication US20140223599, cry proteins such as Cry1A and Cry3A havingmodified proteolytic sites of U.S. Pat. No. 8,319,019; a Cry1Ac, Cry2Aaand Cry1Ca toxin protein from Bacillus thuringiensis strain VBTS 2528 ofUS Patent Application Publication Number 2011/0064710. Other Cryproteins are well known to one skilled in the art (see, Crickmore, etal., “Bacillus thuringiensis toxin nomenclature” (2011), atlifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/ which can be accessed onthe world-wide web using the “www” prefix). The insecticidal activity ofCry proteins is well known to one skilled in the art (for review, see,van Frannkenhuyzen, (2009) J. Invert. Path. 101:1-16). The use of Cryproteins as transgenic plant traits is well known to one skilled in theart and Cry-transgenic plants including but not limited to plantsexpressing Cry1Ac, Cry1Ac+Cry2Ab, Cry1Ab, Cry1A.105, Cry1F, Cry1Fa2,Cry1F+Cry1Ac, Cry2Ab, Cry3A, mCry3A, Cry3Bb1, Cry34Ab1, Cry35Ab1, Vip3A,mCry3A, Cry9c and CBI-Bt have received regulatory approval (see,Sanahuja, (2011) Plant Biotech Journal 9:283-300 and the CERA (2010) GMCrop Database Center for Environmental Risk Assessment (CERA), ILSIResearch Foundation, Washington D.C. atcera-gmc.org/index.php?action=gm_crop_database, which can be accessed onthe world-wide web using the “www” prefix). More than one pesticidalproteins well known to one skilled in the art can also be expressed inplants such as Vip3Ab & Cry1Fa (US2012/0317682); Cry1BE & Cry1F(US2012/0311746); Cry1CA & Cry1AB (US2012/0311745); Cry1F & CryCa(US2012/0317681); Cry1DA & Cry1BE (US2012/0331590); Cry1DA & Cry1Fa(US2012/0331589); Cry1AB & Cry1BE (US2012/0324606); Cry1Fa & Cry2Aa andCry1I & Cry1E (US2012/0324605); Cry34Ab/35Ab and Cry6Aa (US20130167269);Cry34Ab/VCry35Ab & Cry3Aa (US20130167268); Cry1Ab & Cry1F(US20140182018); and Cry3A and Cry1Ab or Vip3Aa (US20130116170).Pesticidal proteins also include insecticidal lipases including lipidacyl hydrolases of U.S. Pat. No. 7,491,869, and cholesterol oxidasessuch as from Streptomyces (Purcell et al. (1993) Biochem Biophys ResCommun 15:1406-1413). Pesticidal proteins also include VIP (vegetativeinsecticidal proteins) toxins of U.S. Pat. Nos. 5,877,012, 6,107,279,6,137,033, 7,244,820, 7,615,686, and 8,237,020, and the like. Other VIPproteins are well known to one skilled in the art (see,lifesci.sussex.ac.uk/home/Neil_Crickmore/Bt/vip.html which can beaccessed on the world-wide web using the “www” prefix). Pesticidalproteins also include toxin complex (TC) proteins, obtainable fromorganisms such as Xenorhabdus, Photorhabdus and Paenibacillus (see, U.S.Pat. Nos. 7,491,698 and 8,084,418). Some TC proteins have “stand alone”insecticidal activity and other TC proteins enhance the activity of thestand-alone toxins produced by the same given organism. The toxicity ofa “stand-alone” TC protein (from Photorhabdus, Xenorhabdus orPaenibacillus, for example) can be enhanced by one or more TC protein“potentiators” derived from a source organism of a different genus.There are three main types of TC proteins. As referred to herein, ClassA proteins (“Protein A”) are stand-alone toxins. Class B proteins(“Protein B”) and Class C proteins (“Protein C”) enhance the toxicity ofClass A proteins. Examples of Class A proteins are TcbA, TcdA, XptA1 andXptA2. Examples of Class B proteins are TcaC, TcdB, XptB1Xb and XptC1Wi.Examples of Class C proteins are TccC, XptC1Xb and XptB1Wi. Pesticidalproteins also include spider, snake and scorpion venom proteins.Examples of spider venom peptides include but are not limited tolycotoxin-1 peptides and mutants thereof (U.S. Pat. No. 8,334,366).

(C) A polynucleotide encoding an insect-specific hormone or pheromonesuch as an ecdysteroid and juvenile hormone, a variant thereof, amimetic based thereon or an antagonist or agonist thereof. See, forexample, the disclosure by Hammock, et al., (1990) Nature 344:458, ofbaculovirus expression of cloned juvenile hormone esterase, aninactivator of juvenile hormone.

(D) A polynucleotide encoding an insect-specific peptide which, uponexpression, disrupts the physiology of the affected pest. For example,see the disclosures of, Regan, (1994) J. Biol. Chem. 269:9 (expressioncloning yields DNA coding for insect diuretic hormone receptor); Pratt,et al., (1989) Biochem. Biophys. Res. Comm. 163:1243 (an allostatin isidentified in Diploptera puntata); Chattopadhyay, et al., (2004)Critical Reviews in Microbiology 30(1):33-54; Zjawiony, (2004) J NatProd 67(2):300-310; Carlini and Grossi-de-Sa, (2002) Toxicon40(11):1515-1539; Ussuf, et al., (2001) Curr Sci. 80(7):847-853 andVasconcelos and Oliveira, (2004) Toxicon 44(4):385-403. See also, U.S.Pat. No. 5,266,317 to Tomalski, et al., who disclose genes encodinginsect-specific toxins.

(E) A polynucleotide encoding an enzyme responsible for ahyperaccumulation of a monoterpene, a sesquiterpene, a steroid,hydroxamic acid, a phenylpropanoid derivative or another non-proteinmolecule with insecticidal activity.

(F) A polynucleotide encoding an enzyme involved in the modification,including the post-translational modification, of a biologically activemolecule; for example, a glycolytic enzyme, a proteolytic enzyme, alipolytic enzyme, a nuclease, a cyclase, a transaminase, an esterase, ahydrolase, a phosphatase, a kinase, a phosphorylase, a polymerase, anelastase, a chitinase and a glucanase, whether natural or synthetic.See, PCT Application WO 1993/02197 in the name of Scott, et al., whichdiscloses the nucleotide sequence of a callase gene. DNA molecules whichcontain chitinase-encoding sequences can be obtained, for example, fromthe ATCC® under Accession Numbers 39637 and 67152. See also, Kramer, etal., (1993) Insect Biochem. Molec. Biol. 23:691, who teach thenucleotide sequence of a cDNA encoding tobacco hookworm chitinase andKawalleck, et al., (1993) Plant Molec. Biol. 21:673, who provide thenucleotide sequence of the parsley ubi4-2 polyubiquitin gene, and U.S.Pat. Nos. 6,563,020; 7,145,060 and 7,087,810.

(G) A polynucleotide encoding a molecule that stimulates signaltransduction. For example, see the disclosure by Botella, et al., (1994)Plant Molec. Biol. 24:757, of nucleotide sequences for mung beancalmodulin cDNA clones, and Griess, et al., (1994) Plant Physiol.104:1467, who provide the nucleotide sequence of a maize calmodulin cDNAclone.

(H) A polynucleotide encoding a hydrophobic moment peptide. See, PCTApplication WO 1995/16776 and U.S. Pat. No. 5,580,852 disclosure ofpeptide derivatives of Tachyplesin which inhibit fungal plant pathogens)and PCT Application WO 1995/18855 and U.S. Pat. No. 5,607,914 (teachessynthetic antimicrobial peptides that confer disease resistance).

(I) A polynucleotide encoding a membrane permease, a channel former or achannel blocker. For example, see the disclosure by Jaynes, et al.,(1993) Plant Sci. 89:43, of heterologous expression of a cecropin-betalytic peptide analog to render transgenic tobacco plants resistant toPseudomonas solanacearum.

(J) A gene encoding a viral-invasive protein or a complex toxin derivedtherefrom. For example, the accumulation of viral coat proteins intransformed plant cells imparts resistance to viral infection and/ordisease development effected by the virus from which the coat proteingene is derived, as well as by related viruses. See, Beachy, et al.,(1990) Ann. Rev. Phytopathol. 28:451. Coat protein-mediated resistancehas been conferred upon transformed plants against alfalfa mosaic virus,cucumber mosaic virus, tobacco streak virus, potato virus X, potatovirus Y, tobacco etch virus, tobacco rattle virus and tobacco mosaicvirus. Id.

(K) A gene encoding an insect-specific antibody or an immunotoxinderived therefrom. Thus, an antibody targeted to a critical metabolicfunction in the insect gut would inactivate an affected enzyme, killingthe insect. Cf. Taylor, et al., Abstract #497, SEVENTH INT'L SYMPOSIUMON MOLECULAR PLANT-MICROBE INTERACTIONS (Edinburgh, Scotland, 1994)(enzymatic inactivation in transgenic tobacco via production ofsingle-chain antibody fragments).

(L) A gene encoding a virus-specific antibody. See, for example,Tavladoraki, et al., (1993) Nature 366:469, who show that transgenicplants expressing recombinant antibody genes are protected from virusattack.

(M) A polynucleotide encoding a developmental-arrestive protein producedin nature by a pathogen or a parasite. Thus, fungal endoalpha-1,4-D-polygalacturonases facilitate fungal colonization and plantnutrient release by solubilizing plant cell wallhomo-alpha-1,4-D-galacturonase. See, Lamb, et al., (1992) Bio/Technology10:1436. The cloning and characterization of a gene which encodes a beanendopolygalacturonase-inhibiting protein is described by Toubart, etal., (1992) Plant J. 2:367.

(N) A polynucleotide encoding a developmental-arrestive protein producedin nature by a plant. For example, Logemann, et al., (1992)Bio/Technology 10:305, have shown that transgenic plants expressing thebarley ribosome-inactivating gene have an increased resistance to fungaldisease.

(O) Genes involved in the Systemic Acquired Resistance (SAR) Responseand/or the pathogenesis related genes. Briggs, (1995) Current Biology5(2), Pieterse and Van Loon, (2004) Curr. Opin. Plant Bio. 7(4):456-64and Somssich, (2003) Cell 113(7):815-6.

(P) Antifungal genes (Cornelissen and Melchers, (1993) Pl. Physiol.101:709-712 and Parijs, et al., (1991) Planta 183:258-264 and Bushnell,et al., (1998) Can. J. of Plant Path. 20(2):137-149. Also see, U.S.patent application Ser. Nos. 09/950,933; 11/619,645; 11/657,710;11/748,994; 11/774,121 and U.S. Pat. Nos. 6,891,085 and 7,306,946. LysMReceptor-like kinases for the perception of chitin fragments as a firststep in plant defense response against fungal pathogens (US2012/0110696).

(Q) Detoxification genes, such as for fumonisin, beauvericin,moniliformin and zearalenone and their structurally related derivatives.For example, see, U.S. Pat. Nos. 5,716,820; 5,792,931; 5,798,255;5,846,812; 6,083,736; 6,538,177; 6,388,171 and 6,812,380.

(R) A polynucleotide encoding a Cystatin and cysteine proteinaseinhibitors. See, U.S. Pat. No. 7,205,453.

(S) Defensin genes. See, WO 2003/000863 and U.S. Pat. Nos. 6,911,577;6,855,865; 6,777,592 and 7,238,781.

(T) Genes conferring resistance to nematodes. See, e.g., PCT ApplicationWO 1996/30517; PCT Application WO 1993/19181, WO 2003/033651 and Urwin,et al., (1998) Planta 204:472-479, Williamson, (1999) Curr Opin PlantBio. 2(4):327-31; U.S. Pat. Nos. 6,284,948 and 7,301,069 and miR164genes (WO 2012/058266).

(U) Genes that confer resistance to Phytophthora Root Rot, such as theRps 1, Rps 1-a, Rps 1-b, Rps 1-c, Rps 1-d, Rps 1-e, Rps 1-k, Rps 2, Rps3-a, Rps 3-b, Rps 3-c, Rps 4, Rps 5, Rps 6, Rps 7 and other Rps genes.See, for example, Shoemaker, et al., Phytophthora Root Rot ResistanceGene Mapping in Soybean, Plant Genome IV Conference, San Diego, Calif.(1995).

(V) Genes that confer resistance to Brown Stem Rot, such as described inU.S. Pat. No. 5,689,035 and incorporated by reference for this purpose.

(W) Genes that confer resistance to Colletotrichum, such as described inUS Patent Application Publication US 2009/0035765 and incorporated byreference for this purpose. This includes the Rcg locus that may beutilized as a single locus conversion.

2. Transgenes that Confer Resistance to a Herbicide, for Example:

(A) A polynucleotide encoding resistance to a herbicide that inhibitsthe growing point or meristem, such as an imidazolinone or asulfonylurea. Exemplary genes in this category code for mutant ALS andAHAS enzyme as described, for example, by Lee, et al., (1988) EMBO J.7:1241 and Miki, et al., (1990) Theor. Appl. Genet. 80:449,respectively. See also, U.S. Pat. Nos. 5,605,011; 5,013,659; 5,141,870;5,767,361; 5,731,180; 5,304,732; 4,761,373; 5,331,107; 5,928,937 and5,378,824; U.S. patent application Ser. No. 11/683,737 and InternationalPublication WO 1996/33270.

(B) A polynucleotide encoding a protein for resistance to Glyphosate(resistance imparted by mutant 5-enolpyruvl-3-phosphikimate synthase(EPSP) and aroA genes, respectively) and other phosphono compounds suchas glufosinate (phosphinothricin acetyl transferase (PAT) andStreptomyces hygroscopicus phosphinothricin acetyl transferase (bar)genes), and pyridinoxy or phenoxy proprionic acids and cyclohexones(ACCase inhibitor-encoding genes). See, for example, U.S. Pat. No.4,940,835 to Shah, et al., which discloses the nucleotide sequence of aform of EPSPS which can confer glyphosate resistance. U.S. Pat. No.5,627,061 to Barry, et al., also describes genes encoding EPSPS enzymes.See also, U.S. Pat. Nos. 6,566,587; 6,338,961; 6,248,876 B1; 6,040,497;5,804,425; 5,633,435; 5,145,783; 4,971,908; 5,312,910; 5,188,642;5,094,945, 4,940,835; 5,866,775; 6,225,114 B1; 6,130,366; 5,310,667;4,535,060; 4,769,061; 5,633,448; 5,510,471; Re. 36,449; RE 37,287 E and5,491,288 and International Publications EP 1173580; WO 2001/66704; EP1173581 and EP 1173582, which are incorporated herein by reference forthis purpose. Glyphosate resistance is also imparted to plants thatexpress a gene encoding a glyphosate oxido-reductase enzyme as describedmore fully in U.S. Pat. Nos. 5,776,760 and 5,463,175, which areincorporated herein by reference for this purpose. In additionglyphosate resistance can be imparted to plants by the over expressionof genes encoding glyphosate N-acetyltransferase. See, for example, U.S.Pat. Nos. 7,462,481; 7,405,074 and US Patent Application PublicationNumber US 2008/0234130. A DNA molecule encoding a mutant aroA gene canbe obtained under ATCC® Accession Number 39256, and the nucleotidesequence of the mutant gene is disclosed in U.S. Pat. No. 4,769,061 toComai. EP Application Number 0 333 033 to Kumada, et al., and U.S. Pat.No. 4,975,374 to Goodman, et al., disclose nucleotide sequences ofglutamine synthetase genes which confer resistance to herbicides such asL-phosphinothricin. The nucleotide sequence of aphosphinothricin-acetyl-transferase gene is provided in EP ApplicationNumbers 0 242 246 and 0 242 236 to Leemans, et al., De Greef, et al.,(1989) Bio/Technology 7:61, describe the production of transgenic plantsthat express chimeric bar genes coding for phosphinothricin acetyltransferase activity. See also, U.S. Pat. Nos. 5,969,213; 5,489,520;5,550,318; 5,874,265; 5,919,675; 5,561,236; 5,648,477; 5,646,024;6,177,616 B1 and 5,879,903, which are incorporated herein by referencefor this purpose. Exemplary genes conferring resistance to phenoxyproprionic acids and cyclohexones, such as sethoxydim and haloxyfop, arethe Acc1-S1, Acc1-S2 and Acc1-S3 genes described by Marshall, et al.,(1992) Theor. Appl. Genet. 83:435.

(C) A polynucleotide encoding a protein for resistance to herbicide thatinhibits photosynthesis, such as a triazine (psbA and gs+ genes) and abenzonitrile (nitrilase gene). Przibilla, et al., (1991) Plant Cell3:169, describe the transformation of Chlamydomonas with plasmidsencoding mutant psbA genes. Nucleotide sequences for nitrilase genes aredisclosed in U.S. Pat. No. 4,810,648 to Stalker and DNA moleculescontaining these genes are available under ATCC® Accession Numbers53435, 67441 and 67442. Cloning and expression of DNA coding for aglutathione S-transferase is described by Hayes, et al., (1992) Biochem.J. 285:173.

(D) A polynucleotide encoding a protein for resistance to Acetohydroxyacid synthase, which has been found to make plants that express thisenzyme resistant to multiple types of herbicides, has been introducedinto a variety of plants (see, e.g., Hattori, et al., (1995) Mol GenGenet. 246:419). Other genes that confer resistance to herbicidesinclude: a gene encoding a chimeric protein of rat cytochrome P4507A1and yeast NADPH-cytochrome P450 oxidoreductase (Shiota, et al., (1994)Plant Physiol 106:17), genes for glutathione reductase and superoxidedismutase (Aono, et al., (1995) Plant Cell Physiol 36:1687) and genesfor various phosphotransferases (Datta, et al., (1992) Plant Mol Biol20:619).

(E) A polynucleotide encoding resistance to a herbicide targetingProtoporphyrinogen oxidase (protox) which is necessary for theproduction of chlorophyll. The protox enzyme serves as the target for avariety of herbicidal compounds. These herbicides also inhibit growth ofall the different species of plants present, causing their totaldestruction. The development of plants containing altered protoxactivity which are resistant to these herbicides are described in U.S.Pat. Nos. 6,288,306 B1; 6,282,837 B1 and 5,767,373 and InternationalPublication WO 2001/12825.

(F) The aad-1 gene (originally from Sphingobium herbicidovorans) encodesthe aryloxyalkanoate dioxygenase (AAD-1) protein. The trait conferstolerance to 2,4-dichlorophenoxyacetic acid and aryloxyphenoxypropionate(commonly referred to as “fop” herbicides such as quizalofop)herbicides. The aad-1 gene, itself, for herbicide tolerance in plantswas first disclosed in WO 2005/107437 (see also, US 2009/0093366). Theaad-12 gene, derived from Delftia acidovorans, which encodes thearyloxyalkanoate dioxygenase (AAD-12) protein that confers tolerance to2,4-dichlorophenoxyacetic acid and pyridyloxyacetate herbicides bydeactivating several herbicides with an aryloxyalkanoate moiety,including phenoxy auxin (e.g., 2,4-D, MCPA), as well as pyridyloxyauxins (e.g., fluroxypyr, triclopyr).

(G) A polynucleotide encoding a herbicide resistant dicambamonooxygenase disclosed in US Patent Application Publication2003/0135879 for imparting dicamba tolerance;

(H) A polynucleotide molecule encoding bromoxynil nitrilase (Bxn)disclosed in U.S. Pat. No. 4,810,648 for imparting bromoxynil tolerance;

(I) A polynucleotide molecule encoding phytoene (crtl) described inMisawa, et al., (1993) Plant J. 4:833-840 and in Misawa, et al., (1994)Plant J. 6:481-489 for norflurazon tolerance.

3. Transgenes that Confer or Contribute to an Altered GrainCharacteristic

Such as:

(A) Altered fatty acids, for example, by

(1) Down-regulation of stearoyl-ACP to increase stearic acid content ofthe plant. See, Knultzon, et al., (1992) Proc. Natl. Acad. Sci. USA89:2624 and WO 1999/64579 (Genes to Alter Lipid Profiles in Corn).

(2) Elevating oleic acid via FAD-2 gene modification and/or decreasinglinolenic acid via FAD-3 gene modification (see, U.S. Pat. Nos.6,063,947; 6,323,392; 6,372,965 and WO 1993/11245).

(3) Altering conjugated linolenic or linoleic acid content, such as inWO 2001/12800.

(4) Altering LEC1, AGP, Dek1, Superal1, mi1 ps, various Ipa genes suchas Ipa1, Ipa3, hpt or hggt. For example, see, WO 2002/42424, WO1998/22604, WO 2003/011015, WO 2002/057439, WO 2003/011015, U.S. Pat.Nos. 6,423,886, 6,197,561, 6,825,397 and US Patent ApplicationPublication Numbers US 2003/0079247, US 2003/0204870 and Rivera-Madrid,et al., (1995) Proc. Natl. Acad. Sci. 92:5620-5624.

(5) Genes encoding delta-8 desaturase for making long-chainpolyunsaturated fatty acids (U.S. Pat. Nos. 8,058,571 and 8,338,152),delta-9 desaturase for lowering saturated fats (U.S. Pat. No.8,063,269), Primula A6-desaturase for improving omega-3 fatty acidprofiles.

(6) Isolated nucleic acids and proteins associated with lipid and sugarmetabolism regulation, in particular, lipid metabolism protein (LMP)used in methods of producing transgenic plants and modulating levels ofseed storage compounds including lipids, fatty acids, starches or seedstorage proteins and use in methods of modulating the seed size, seednumber, seed weights, root length and leaf size of plants (EP 2404499).

(7) Altering expression of a High-Level Expression of Sugar-Inducible 2(HSI2) protein in the plant to increase or decrease expression of HSI2in the plant. Increasing expression of HSI2 increases oil content whiledecreasing expression of HSI2 decreases abscisic acid sensitivity and/orincreases drought resistance (US Patent Application Publication Number2012/0066794).

(8) Expression of cytochrome b5 (Cb5) alone or with FAD2 to modulate oilcontent in plant seed, particularly to increase the levels of omega-3fatty acids and improve the ratio of omega-6 to omega-3 fatty acids (USPatent Application Publication Number 2011/0191904).

(9) Nucleic acid molecules encoding wrinkled1-like polypeptides formodulating sugar metabolism (U.S. Pat. No. 8,217,223).

(B) Altered phosphorus content, for example, by the

(1) Introduction of a phytase-encoding gene would enhance breakdown ofphytate, adding more free phosphate to the transformed plant. Forexample, see, Van Hartingsveldt, et al., (1993) Gene 127:87, for adisclosure of the nucleotide sequence of an Aspergillus niger phytasegene.

(2) Modulating a gene that reduces phytate content. In maize, this, forexample, could be accomplished, by cloning and then re-introducing DNAassociated with one or more of the alleles, such as the LPA alleles,identified in maize mutants characterized by low levels of phytic acid,such as in WO 2005/113778 and/or by altering inositol kinase activity asin WO 2002/059324, US Patent Application Publication Number2003/0009011, WO 2003/027243, US Patent Application Publication Number2003/0079247, WO 1999/05298, U.S. Pat. Nos. 6,197,561, 6,291,224,6,391,348, WO 2002/059324, US Patent Application Publication Number2003/0079247, WO 1998/45448, WO 1999/55882, WO 2001/04147.

(C) Altered carbohydrates affected, for example, by altering a gene foran enzyme that affects the branching pattern of starch or, a genealtering thioredoxin such as NTR and/or TRX (see, U.S. Pat. No.6,531,648. which is incorporated by reference for this purpose) and/or agamma zein knock out or mutant such as cs27 or TUSC27 or en27 (see, U.S.Pat. No. 6,858,778 and US Patent Application Publication Number2005/0160488, US Patent Application Publication Number 2005/0204418,which are incorporated by reference for this purpose). See, Shiroza, etal., (1988) J. Bacteriol. 170:810 (nucleotide sequence of Streptococcusmutant fructosyltransferase gene), Steinmetz, et al., (1985) Mol. Gen.Genet. 200:220 (nucleotide sequence of Bacillus subtilis levansucrasegene), Pen, et al., (1992) Bio/Technology 10:292 (production oftransgenic plants that express Bacillus licheniformis alpha-amylase),Elliot, et al., (1993) Plant Molec. Biol. 21:515 (nucleotide sequencesof tomato invertase genes), Segaard, et al., (1993) J. Biol. Chem.268:22480 (site-directed mutagenesis of barley alpha-amylase gene) andFisher, et al., (1993) Plant Physiol. 102:1045 (maize endosperm starchbranching enzyme II), WO 1999/10498 (improved digestibility and/orstarch extraction through modification of UDP-D-xylose 4-epimerase,Fragile 1 and 2, Ref1, HCHL, C4H), U.S. Pat. No. 6,232,529 (method ofproducing high oil seed by modification of starch levels (AGP)). Thefatty acid modification genes mentioned herein may also be used toaffect starch content and/or composition through the interrelationshipof the starch and oil pathways.

(D) Altered antioxidant content or composition, such as alteration oftocopherol or tocotrienols. For example, see, U.S. Pat. No. 6,787,683,US Patent Application Publication Number 2004/0034886 and WO 2000/68393involving the manipulation of antioxidant levels and WO 2003/082899through alteration of a homogentisate geranyl geranyl transferase(hggt).

(E) Altered essential seed amino acids. For example, see, U.S. Pat. No.6,127,600 (method of increasing accumulation of essential amino acids inseeds), U.S. Pat. No. 6,080,913 (binary methods of increasingaccumulation of essential amino acids in seeds), U.S. Pat. No. 5,990,389(high lysine), WO 1999/40209 (alteration of amino acid compositions inseeds), WO 1999/29882 (methods for altering amino acid content ofproteins), U.S. Pat. No. 5,850,016 (alteration of amino acidcompositions in seeds), WO 1998/20133 (proteins with enhanced levels ofessential amino acids), U.S. Pat. No. 5,885,802 (high methionine), U.S.Pat. No. 5,885,801 (high threonine), U.S. Pat. No. 6,664,445 (plantamino acid biosynthetic enzymes), U.S. Pat. No. 6,459,019 (increasedlysine and threonine), U.S. Pat. No. 6,441,274 (plant tryptophansynthase beta subunit), U.S. Pat. No. 6,346,403 (methionine metabolicenzymes), U.S. Pat. No. 5,939,599 (high sulfur), U.S. Pat. No. 5,912,414(increased methionine), WO 1998/56935 (plant amino acid biosyntheticenzymes), WO 1998/45458 (engineered seed protein having higherpercentage of essential amino acids), WO 1998/42831 (increased lysine),U.S. Pat. No. 5,633,436 (increasing sulfur amino acid content), U.S.Pat. No. 5,559,223 (synthetic storage proteins with defined structurecontaining programmable levels of essential amino acids for improvementof the nutritional value of plants), WO 1996/01905 (increasedthreonine), WO 1995/15392 (increased lysine), US Patent ApplicationPublication Number 2003/0163838, US Patent Application PublicationNumber 2003/0150014, US Patent Application Publication Number2004/0068767, U.S. Pat. No. 6,803,498, WO 2001/79516.

4. Genes that Control Male-Sterility:

There are several methods of conferring genetic male sterilityavailable, such as multiple mutant genes at separate locations withinthe genome that confer male sterility, as disclosed in U.S. Pat. Nos.4,654,465 and 4,727,219 to Brar, et al., and chromosomal translocationsas described by Patterson in U.S. Pat. Nos. 3,861,709 and 3,710,511. Inaddition to these methods, Albertsen, et al., U.S. Pat. No. 5,432,068,describe a system of nuclear male sterility which includes: identifyinga gene which is critical to male fertility; silencing this native genewhich is critical to male fertility; removing the native promoter fromthe essential male fertility gene and replacing it with an induciblepromoter; inserting this genetically engineered gene back into theplant; and thus creating a plant that is male sterile because theinducible promoter is not “on” resulting in the male fertility gene notbeing transcribed. Fertility is restored by inducing or turning “on”,the promoter, which in turn allows the gene that confers male fertilityto be transcribed.

(A) Introduction of a deacetylase gene under the control of atapetum-specific promoter and with the application of the chemicalN—Ac-PPT (WO 2001/29237).

(B) Introduction of various stamen-specific promoters (WO 1992/13956, WO1992/13957).

(C) Introduction of the barnase and the barstar gene (Paul, et al.,(1992) Plant Mol. Biol. 19:611-622).

For additional examples of nuclear male and female sterility systems andgenes, see also, U.S. Pat. Nos. 5,859,341; 6,297,426; 5,478,369;5,824,524; 5,850,014 and 6,265,640, all of which are hereby incorporatedby reference.

5. Genes that Create a Site for Site Specific DNA Integration.

This includes the introduction of FRT sites that may be used in theFLP/FRT system and/or Lox sites that may be used in the Cre/Loxp system.For example, see, Lyznik, et al., (2003) Plant Cell Rep 21:925-932 andWO 1999/25821, which are hereby incorporated by reference. Other systemsthat may be used include the Gln recombinase of phage Mu (Maeser, etal., (1991) Vicki Chandler, The Maize Handbook ch. 118 (Springer-Verlag1994), the Pin recombinase of E. coli (Enomoto, et al., 1983) and theR/RS system of the pSRi plasmid (Araki, et al., 1992).

6. Genes that Affect Abiotic Stress Resistance

Including but not limited to flowering, ear and seed development,enhancement of nitrogen utilization efficiency, altered nitrogenresponsiveness, drought resistance or tolerance, cold resistance ortolerance and salt resistance or tolerance and increased yield understress.

(A) For example, see: WO 2000/73475 where water use efficiency isaltered through alteration of malate; U.S. Pat. Nos. 5,892,009,5,965,705, 5,929,305, 5,891,859, 6,417,428, 6,664,446, 6,706,866,6,717,034, 6,801,104, WO 2000/060089, WO 2001/026459, WO 2001/035725, WO2001/034726, WO 2001/035727, WO 2001/036444, WO 2001/036597, WO2001/036598, WO 2002/015675, WO 2002/017430, WO 2002/077185, WO2002/079403, WO 2003/013227, WO 2003/013228, WO 2003/014327, WO2004/031349, WO 2004/076638, WO 199809521.

(B) WO 199938977 describing genes, including CBF genes and transcriptionfactors effective in mitigating the negative effects of freezing, highsalinity and drought on plants, as well as conferring other positiveeffects on plant phenotype.

(C) US Patent Application Publication Number 2004/0148654 and WO2001/36596 where abscisic acid is altered in plants resulting inimproved plant phenotype such as increased yield and/or increasedtolerance to abiotic stress.

(D) WO 2000/006341, WO 2004/090143, U.S. Pat. Nos. 7,531,723 and6,992,237 where cytokinin expression is modified resulting in plantswith increased stress tolerance, such as drought tolerance, and/orincreased yield. Also see, WO 2002/02776, WO 2003/052063, JP2002/281975, U.S. Pat. No. 6,084,153, WO 2001/64898, U.S. Pat. Nos.6,177,275 and 6,107,547 (enhancement of nitrogen utilization and alterednitrogen responsiveness).

(E) For ethylene alteration, see, US Patent Application PublicationNumber 2004/0128719, US Patent Application Publication Number2003/0166197 and WO 2000/32761.

(F) For plant transcription factors or transcriptional regulators ofabiotic stress, see, e.g., US Patent Application Publication Number2004/0098764 or US Patent Application Publication Number 2004/0078852.

(G) Genes that increase expression of vacuolar pyrophosphatase such asAVP1 (U.S. Pat. No. 8,058,515) for increased yield; nucleic acidencoding a HSFA4 or a HSFA5 (Heat Shock Factor of the class A4 or A5)polypeptides, an oligopeptide transporter protein (OPT4-like)polypeptide; a plastochron2-like (PLA2-like) polypeptide or a Wuschelrelated homeobox 1-like (WOX1-like) polypeptide (U. Patent ApplicationPublication Number US 2011/0283420).

(H) Down regulation of polynucleotides encoding poly (ADP-ribose)polymerase (PARP) proteins to modulate programmed cell death (U.S. Pat.No. 8,058,510) for increased vigor.

(I) Polynucleotide encoding DTP21 polypeptides for conferring droughtresistance (US Patent Application Publication Number US 2011/0277181).

(J) Nucleotide sequences encoding ACC Synthase 3 (ACS3) proteins formodulating development, modulating response to stress, and modulatingstress tolerance (US Patent Application Publication Number US2010/0287669).

(K) Polynucleotides that encode proteins that confer a drought tolerancephenotype (DTP) for conferring drought resistance (WO 2012/058528).

(L) Tocopherol cyclase (TC) genes for conferring drought and salttolerance (US Patent Application Publication Number 2012/0272352).

(M) CAAX amino terminal family proteins for stress tolerance (U.S. Pat.No. 8,338,661).

(N) Mutations in the SAL1 encoding gene have increased stress tolerance,including increased drought resistant (US Patent Application PublicationNumber 2010/0257633).

(O) Expression of a nucleic acid sequence encoding a polypeptideselected from the group consisting of: GRF polypeptide, RAA1-likepolypeptide, SYR polypeptide, ARKL polypeptide, and YTP polypeptideincreasing yield-related traits (US Patent Application PublicationNumber 2011/0061133).

(P) Modulating expression in a plant of a nucleic acid encoding a ClassIII Trehalose Phosphate Phosphatase (TPP) polypeptide for enhancingyield-related traits in plants, particularly increasing seed yield (USPatent Application Publication Number 2010/0024067).

(Q) Expression of a nucleic acid sequence encoding a Drought TolerantPhenotype (DTP6) polypeptide, specifically AT-DTP6 of US PatentApplication Publication Number US-2014/0223595.

Other genes and transcription factors that affect plant growth andagronomic traits such as yield, flowering, plant growth and/or plantstructure, can be introduced or introgressed into plants, see e.g., WO1997/49811 (LHY), WO 1998/56918 (ESD4), WO 1997/10339 and U.S. Pat. No.6,573,430 (TFL), U.S. Pat. No. 6,713,663 (FT), WO 1996/14414 (CON), WO1996/38560, WO 2001/21822 (VRN1), WO 2000/44918 (VRN2), WO 1999/49064(GI), WO 2000/46358 (FR1), WO 1997/29123, U.S. Pat. Nos. 6,794,560,6,307,126 (GAI), WO 1999/09174 (D8 and Rht) and WO 2004/076638 and WO2004/031349 (transcription factors).

7. Genes that Confer Increased Yield

(A) A transgenic crop plant transformed by a1-AminoCyclopropane-1-Carboxylate Deaminase-like Polypeptide (ACCDP)coding nucleic acid, wherein expression of the nucleic acid sequence inthe crop plant results in the plant's increased root growth, and/orincreased yield, and/or increased tolerance to environmental stress ascompared to a wild type variety of the plant (U.S. Pat. No. 8,097,769).

(B) Over-expression of maize zinc finger protein gene (Zm-ZFP1) using aseed preferred promoter has been shown to enhance plant growth, increasekernel number and total kernel weight per plant (US Patent ApplicationPublication Number 2012/0079623).

(C) Constitutive over-expression of maize lateral organ boundaries (LOB)domain protein (Zm-LOBDP1) has been shown to increase kernel number andtotal kernel weight per plant (US Patent Application Publication Number2012/0079622).

(D) Enhancing yield-related traits in plants by modulating expression ina plant of a nucleic acid encoding a VIM1 (Variant in Methylation1)-like polypeptide or a VTC2-like (GDP-L-galactose phosphorylase)polypeptide or a DUF1685 polypeptide or an ARF6-like (Auxin ResponsiveFactor) polypeptide (WO 2012/038893).

(E) Modulating expression in a plant of a nucleic acid encoding aSte20-like polypeptide or a homologue thereof gives plants havingincreased yield relative to control plants (EP 2431472).

(F) Genes encoding nucleoside diphosphatase kinase (NDK) polypeptidesand homologs thereof for modifying the plant's root architecture (USPatent Application Publication Number 2009/0064373).

8. Genes that Confer Plant Digestibility.

(A) Altering the level of xylan present in the cell wall of a plant bymodulating expression of xylan synthase (U.S. Pat. No. 8,173,866).

In some embodiment the stacked trait may be a trait or event that hasreceived regulatory approval including but not limited to the events inTable 4A-4F.

TABLE 4A Oryza sativa Rice Event Company Description CL121, CL141, CFX51BASF Inc. Tolerance to the imidazolinone herbicide, imazethapyr, inducedby chemical mutagenesis of the acetolactate synthase (ALS) enzyme usingethyl methanesulfonate (EMS). IMINTA-1, IMINTA-4 BASF Inc. Tolerance toimidazolinone herbicides induced by chemical mutagenesis of theacetolactate synthase (ALS) enzyme using sodium azide. LLRICE06,LLRICE62 Aventis CropScience Glufosinate ammonium herbicide tolerantrice produced by inserting a modified phosphinothricin acetyltransferase(PAT) encoding gene from the soil bacterium Streptomyces hygroscopicus).LLRICE601 Bayer CropScience Glufosinate ammonium herbicide tolerant rice(Aventis produced by inserting a modified CropScience(AgrEvo))phosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus). PWC16 BASF Inc. Tolerance to theimidazolinone herbicide, imazethapyr, induced by chemical mutagenesis ofthe acetolactate synthase (ALS) enzyme using ethyl methanesulfonate(EMS).

TABLE 4B Medicago sativa Alfalfa Event Company Description J101, J163Monsanto Company Glyphosate herbicide tolerant and Forage Geneticsalfalfa (lucerne) produced by International inserting a gene encodingthe enzyme 5-enolypyruvylshikimate- 3-phosphate synthase (EPSPS) fromthe CP4 strain of Agrobacterium tumefaciens.

TABLE 4C Triticum aestivum Wheat Event Company Description AP205CL BASFInc. Selection for a mutagenized version of the enzyme acetohydroxyacidsynthase (AHAS), also known as acetolactate synthase (ALS) oracetolactate pyruvate-lyase. AP602CL BASF Inc. Selection for amutagenized version of the enzyme acetohydroxyacid synthase (AHAS), alsoknown as acetolactate synthase (ALS) or acetolactate pyruvate-lyase.BW255-2, BW238-3 BASF Inc. Selection for a mutagenized version of theenzyme acetohydroxyacid synthase (AHAS), also known as acetolactatesynthase (ALS) or acetolactate pyruvate-lyase. BW7 BASF Inc. Toleranceto imidazolinone herbicides induced by chemical mutagenesis of theacetohydroxyacid synthase (AHAS) gene using sodium azide. MON71800Monsanto Company Glyphosate tolerant wheat variety produced by insertinga modified 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encodinggene from the soil bacterium Agrobacterium tumefaciens, strain CP4.SWP965001 Cyanamid Crop Selection for a mutagenized version of theProtection enzyme acetohydroxyacid synthase (AHAS), also known asacetolactate synthase (ALS) or acetolactate pyruvate-lyase. Teal 11ABASF Inc. Selection for a mutagenized version of the enzymeacetohydroxyacid synthase (AHAS), also known as acetolactate synthase(ALS) or acetolactate pyruvate-lyase.

TABLE 4D Helianthus annuus Sunflower Event Company Description X81359BASF Inc. Tolerance to imidazolinone herbicides by selection of anaturally occurring mutant.

TABLE 4E Glycine max L. Soybean Event Company Description A2704-12,A2704-21, Bayer CropScience Glufosinate ammonium herbicide tolerantA5547-35 (Aventis CropScience soybean produced by inserting a modified(AgrEvo)) phosphinothricin acetyltransferase (PAT) encoding gene fromthe soil bacterium Streptomyces viridochromogenes. A5547-127 BayerCropScience Glufosinate ammonium herbicide tolerant (Aventis CropSciencesoybean produced by inserting a modified (AgrEvo)) phosphinothricinacetyltransferase (PAT) encoding gene from the soil bacteriumStreptomyces viridochromogenes. BPS-CV127-9 BASF Inc. The introducedcsr1-2 gene from Arabidopsis thaliana encodes an acetohydroxyacidsynthase protein that confers tolerance to imidazolinone herbicides dueto a point mutation that results in a single amino acid substitution inwhich the serine residue at position 653 is replaced by asparagine(S653N). DP-305423 Pioneer Hi-Bred High oleic acid soybean produced byinserting International Inc. additional copies of a portion of theomega-6 desaturase encoding gene, gm-fad2-1 resulting in silencing ofthe endogenous omega-6 desaturase gene (FAD2-1). DP356043 PioneerHi-Bred Soybean event with two herbicide tolerance International Inc.genes: glyphosate N-acetlytransferase, which detoxifies glyphosate, anda modified acetolactate synthase (ALS) gene which is tolerant toALS-inhibiting herbicides. G94-1, G94-19, G168 DuPont Canada High oleicacid soybean produced by inserting a Agricultural Products second copyof the fatty acid desaturase (Gm Fad2-1) encoding gene from soybean,which resulted in “silencing” of the endogenous host gene. GTS 40-3-2Monsanto Company Glyphosate tolerant soybean variety produced byinserting a modified 5-enolpyruvylshikimate-3- phosphate synthase(EPSPS) encoding gene from the soil bacterium Agrobacterium tumefaciens.GU262 Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventissoybean produced by inserting a modified CropScience(AgrEvo))phosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces viridochromogenes. MON87701 Monsanto CompanyResistance to Lepidopteran pests of soybean including velvetbeancaterpillar (Anticarsia gemmatalis) and soybean looper (Pseudoplusiaincludens). MON87701 × Monsanto Company Glyphosate herbicide tolerancethrough MON89788 expression of the EPSPS encoding gene from A.tumefaciens strain CP4, and resistance to Lepidopteran pests of soybeanincluding velvetbean caterpillar (Anticarsia gemmatalis) and soybeanlooper (Pseudoplusia includens) via expression of the Cry1Ac encodinggene from B. thuringiensis. MON89788 Monsanto CompanyGlyphosate-tolerant soybean produced by inserting a modified5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) encoding aroA(epsps) gene from Agrobacterium tumefaciens CP4. OT96-15 Agriculture &Agri-Food Low linolenic acid soybean produced through Canada traditionalcross-breeding to incorporate the novel trait from a naturally occurringfan1 gene mutant that was selected for low linolenic acid. W62, W98Bayer CropScience Glufosinate ammonium herbicide tolerant (Aventissoybean produced by inserting a modified CropScience(AgrEvo))phosphinothricin acetyltransferase (PAT) encoding gene from the soilbacterium Streptomyces hygroscopicus.

TABLE 4F Zea mays L. Maize Event Company Description 176 Syngenta Seeds,Inc. Insect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). 3751 IR PioneerHi-Bred Selection of somaclonal variants by culture of InternationalInc. embryos on imidazolinone containing media. 676, 678, 680 PioneerHi-Bred Male-sterile and glufosinate ammonium herbicide InternationalInc. tolerant maize produced by inserting genes encoding DNA adeninemethylase and phosphinothricin acetyltransferase (PAT) from Escherichiacoli and Streptomyces viridochromogenes, respectively. B16 (DLL25)Dekalb Genetics Glufosinate ammonium herbicide tolerant maizeCorporation produced by inserting the gene encoding phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 (X4334CBR,Syngenta Seeds, Inc. Insect-resistant and herbicide tolerant maizeX4734CBR) produced by inserting the Cry1Ab gene from Bacillusthuringiensis subsp. kurstaki, and the phosphinothricinN-acetyltransferase (PAT) encoding gene from S. viridochromogenes. BT11× GA21 Syngenta Seeds, Inc. Stacked insect resistant and herbicidetolerant maize produced by conventional cross breeding of parental linesBT11 (OECD unique identifier: SYN-BTO11-1) and GA21 (OECD uniqueidentifier: MON-OOO21-9). BT11 × MIR162 × Syngenta Seeds, Inc.Resistance to Coleopteran pests, particularly MIR604 × GA21 cornrootworm pests (Diabrotica spp.) and several Lepidopteran pests of corn,including European corn borer (ECB, Ostrinia nubilalis), corn earworm(CEW, Helicoverpa zea), fall army worm (FAW, Spodoptera frugiperda), andblack cutworm (BCW, Agrotis ipsilon); tolerance to glyphosate andglufosinate-ammonium containing herbicides. BT11 × MIR162 SyngentaSeeds, Inc. Stacked insect resistant and herbicide tolerant maizeproduced by conventional cross breeding of parental lines BT11 (OECDunique identifier: SYN-BTO11-1) and MIR162 (OECD unique identifier:SYN-1R162-4). Resistance to the European Corn Borer and tolerance to theherbicide glufosinate ammonium (Liberty) is derived from BT11, whichcontains the Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki,and the phosphinothricin N-acetyltransferase (PAT) encoding gene from S.viridochromogenes. Resistance to other Lepidopteran pests, including H.zea, S. frugiperda, A. ipsilon, and S. albicosta, is derived fromMIR162, which contains the vip3Aa gene from Bacillus thuringiensisstrain AB88. BT11 × MIR162 × Syngenta Seeds, Inc. Bacillus thuringiensisCry1Ab delta-endotoxin MIR604 protein and the genetic material necessaryfor its production (via elements of vector pZO1502) in Event Bt11 corn(OECD Unique Identifier: SYN- BTO11-1) × Bacillus thuringiensis Vip3Aa20insecticidal protein and the genetic material necessary for itsproduction (via elements of vector pNOV1300) in Event MIR162 maize (OECDUnique Identifier: SYN-IR162-4) × modified Cry3A protein and the geneticmaterial necessary for its production (via elements of vector pZM26) inEvent MIR604 corn (OECD Unique Identifier: SYN-1R604-5). CBH-351 AventisCropScience Insect-resistant and glufosinate ammonium herbicide tolerantmaize developed by inserting genes encoding Cry9C protein from Bacillusthuringiensis subsp tolworthi and phosphinothricin acetyltransferase(PAT) from Streptomyces hygroscopicus. DAS-06275-8 DOW AgroSciencesLepidopteran insect resistant and glufosinate LLC ammoniumherbicide-tolerant maize variety produced by inserting the Cry1F genefrom Bacillus thuringiensis var aizawai and the phosphinothricinacetyltransferase (PAT) from Streptomyces hygroscopicus. BT11 × MIR604Syngenta Seeds, Inc. Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines BT11(OECD unique identifier: SYN-BTO11-1) and MIR604 (OECD uniqueidentifier: SYN-1R6O5-5). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm -resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. BT11 × MIR604 × GA21 Syngenta Seeds, Inc. Stacked insectresistant and herbicide tolerant maize produced by conventional crossbreeding of parental lines BT11 (OECD unique identifier: SYN-BTO11-1),MIR604 (OECD unique identifier: SYN-1R6O5-5) and GA21 (OECD uniqueidentifier: MON-OOO21-9). Resistance to the European Corn Borer andtolerance to the herbicide glufosinate ammonium (Liberty) is derivedfrom BT11, which contains the Cry1Ab gene from Bacillus thuringiensissubsp. kurstaki, and the phosphinothricin N-acetyltransferase (PAT)encoding gene from S. viridochromogenes. Corn rootworm-resistance isderived from MIR604 which contains the mCry3A gene from Bacillusthuringiensis. Tolerance to glyphosate herbicide is derived from GA21which contains a a modified EPSPS gene from maize. DAS-59122-7 DOWAgroSciences Corn rootworm-resistant maize produced by LLC and PioneerHi- inserting the Cry34Ab1 and Cry35Ab1 genes Bred International Inc.from Bacillus thuringiensis strain PS149B1. The PAT encoding gene fromStreptomyces viridochromogenes was introduced as a selectable marker.DAS-59122-7 × DOW AgroSciences Stacked insect resistant and herbicidetolerant TC1507 × NK603 LLC and Pioneer Hi- maize produced byconventional cross breeding Bred International Inc. of parental linesDAS-59122-7 (OECD unique identifier: DAS-59122-7) and TC1507 (OECDunique identifier: DAS-O1507-1) with NK603 (OECD unique identifier:MON-OO6O3-6). Corn rootworm-resistance is derived from DAS-59122- 7which contains the Cry34Ab1 and Cry35Ab1 genes from Bacillusthuringiensis strain P5149B1. Lepidopteran resistance and tolerance toglufosinate ammonium herbicide is derived from TC1507. Tolerance toglyphosate herbicide is derived from NK603. DBT418 Dekalb GeneticsInsect-resistant and glufosinate ammonium Corporation herbicide tolerantmaize developed by inserting genes encoding Cry1AC protein from Bacillusthuringiensis subsp kurstaki and phosphinothricin acetyltransferase(PAT) from Streptomyces hygroscopicus MIR604 × GA21 Syngenta Seeds, Inc.Stacked insect resistant and herbicide tolerant maize produced byconventional cross breeding of parental lines MIR604 (OECD uniqueidentifier: SYN-1R6O5-5) and GA21 (OECD unique identifier: MON-OOO21-9).Corn rootworm-resistance is derived from MIR604 which contains themCry3A gene from Bacillus thuringiensis. Tolerance to glyphosateherbicide is derived from GA21. MON80100 Monsanto CompanyInsect-resistant maize produced by inserting the Cry1Ab gene fromBacillus thuringiensis subsp. kurstaki. The genetic modification affordsresistance to attack by the European corn borer (ECB). MON802 MonsantoCompany Insect-resistant and glyphosate herbicide tolerant maizeproduced by inserting the genes encoding the Cry1Ab protein fromBacillus thuringiensis and the 5-enolpyruvylshikimate-3-phosphatesynthase (EPSPS) from A. tumefaciens strain CP4. MON809 Pioneer Hi-BredResistance to European corn borer (Ostrinia International Inc.nubilalis) by introduction of a synthetic Cry1Ab gene. Glyphosateresistance via introduction of the bacterial version of a plant enzyme,5- enolpyruvyl shikimate-3-phosphate synthase (EPSPS). MON810 MonsantoCompany Insect-resistant maize produced by inserting a truncated form ofthe Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1. Thegenetic modification affords resistance to attack by the European cornborer (ECB). MON810 × LY038 Monsanto Company Stacked insect resistantand enhanced lysine content maize derived from conventional cross-breeding of the parental lines MON810 (OECD identifier: MON-OO81O-6) andLY038 (OECD identifier: REN-OOO38-3). MON810 × MON88017 Monsanto CompanyStacked insect resistant and glyphosate tolerant maize derived fromconventional cross-breeding of the parental lines MON810 (OECDidentifier: MON-OO81O-6) and M0N88017 (OECD identifier: MON-88017-3).European corn borer (ECB) resistance is derived from a truncated form ofthe Cry1Ab gene from Bacillus thuringiensis subsp. kurstaki HD-1 presentin MON810. Corn rootworm resistance is derived from the Cry3Bb1 genefrom Bacillus thuringiensis subspecies kumamotoensis strain EG4691present in MON88017. Glyphosate tolerance is derived from a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4 present in MON88017. MON832Monsanto Company Introduction, by particle bombardment, of glyphosateoxidase (GOX) and a modified 5- enolpyruvyl shikimate-3-phosphatesynthase (EPSPS), an enzyme involved in the shikimate biochemicalpathway for the production of the aromatic amino acids. MON863 MonsantoCompany Corn rootworm resistant maize produced by inserting the Cry3Bb1gene from Bacillus thuringiensis subsp. kumamotoensis. MON863 × MON810Monsanto Company Stacked insect resistant corn hybrid derived fromconventional cross-breeding of the parental lines MON863 (OECDidentifier: MON-OO863-5) and MON810 (OECD identifier: MON-OO81O-6)MON863 × MON810 × Monsanto Company Stacked insect resistant andherbicide tolerant NK603 corn hybrid derived from conventional cross-breeding of the stacked hybrid MON-OO863-5 × MON-OO81O-6 and NK603 (OECDidentifier: MON-OO6O3-6). MON863 × NK603 Monsanto Company Stacked insectresistant and herbicide tolerant corn hybrid derived from conventionalcross- breeding of the parental lines MON863 (OECD identifier:MON-OO863-5) and NK603 (OECD identifier: MON-OO6O3-6). MON87460 MonsantoCompany MON 87460 was developed to provide reduced yield loss underwater-limited conditions compared to conventional maize. Efficacy in MON87460 is derived by expression of the inserted Bacillus subtilis coldshock protein B (CspB). MON88017 Monsanto Company Cornrootworm-resistant maize produced by inserting the Cry3Bb1 gene fromBacillus thuringiensis subspecies kumamotoensis strain EG4691.Glyphosate tolerance derived by inserting a5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene fromAgrobacterium tumefaciens strain CP4. MON89034 Monsanto Company Maizeevent expressing two different insecticidal proteins from Bacillusthuringiensis providing resistance to number of Lepidopteran pests.MON89034 × Monsanto Company Stacked insect resistant and glyphosatetolerant MON88017 maize derived from conventional cross-breeding of theparental lines MON89034 (OECD identifier: MON-89O34-3) and MON88017(OECD identifier: MON-88O17-3). Resistance to Lepidopteran insects isderived from two Cry genes present in MON89043. Corn rootworm resistanceis derived from a single Cry genes and glyphosate tolerance is derivedfrom the 5- enolpyruvylshikimate-3-phosphate synthase (EPSPS) encodinggene from Agrobacterium tumefaciens present in MON88017. MON89034 ×NK603 Monsanto Company Stacked insect resistant and herbicide tolerantmaize produced by conventional cross breeding of parental lines MON89034(OECD identifier: MON-89034-3) with NK603 (OECD unique identifier:MON-OO6O3-6). Resistance to Lepidopteran insects is derived from two Crygenes present in MON89043. Tolerance to glyphosate herbicide is derivedfrom NK603. NK603 × MON810 Monsanto Company Stacked insect resistant andherbicide tolerant corn hybrid derived from conventional cross- breedingof the parental lines NK603 (OECD identifier: MON-OO6O3-6) and MON810(OECD identifier: MON-OO81O-6). MON89034 × TC1507 × Monsanto CompanyStacked insect resistant and herbicide tolerant MON88017 × DAS- andMycogen Seeds c/o maize produced by conventional cross breeding 59122-7Dow AgroSciences LLC of parental lines: MON89034, TC1507, MON88017, andDAS-59122. Resistance to the above-ground and below-ground insect pestsand tolerance to glyphosate and glufosinate- ammonium containingherbicides. M53 Bayer CropScience Male sterility caused by expression ofthe (Aventis barnase ribonuclease gene from BacillusCropScience(AgrEvo)) amyloliquefaciens; PPT resistance was via PPT-acetyltransferase (PAT). M56 Bayer CropScience Male sterility caused byexpression of the (Aventis barnase ribonuclease gene from BacillusCropScience(AgrEvo)) amyloliquefaciens; PPT resistance was via PPT-acetyltransferase (PAT). NK603 Monsanto Company Introduction, byparticle bombardment, of a modified 5-enolpyruvyl shikimate-3-phosphatesynthase (EPSPS), an enzyme involved in the shikim ate biochemicalpathway for the production of the aromatic amino acids. NK603 × T25Monsanto Company Stacked glufosinate ammonium and glyphosate herbicidetolerant maize hybrid derived from conventional cross-breeding of theparental lines NK603 (OECD identifier: MON-OO6O3-6) and T25 (OECDidentifier: ACS-ZM003-2). T25 × MON810 Bayer CropScience Stacked insectresistant and herbicide tolerant (Aventis corn hybrid derived fromconventional cross- CropScience(AgrEvo)) breeding of the parental linesT25 (OECD identifier: ACS-ZMOO3-2) and MON810 (OECD identifier:MON-OO81O-6). TC1507 Mycogen (c/o Dow Insect-resistant and glufosinateammonium AgroSciences); Pioneer herbicide tolerant maize produced byinserting (c/o DuPont) the Cry1F gene from Bacillus thuringiensis var.aizawai and the phosphinothricin N- acetyltransferase encoding gene fromStreptomyces viridochromogenes. TC1507 × NK603 DOW AgroSciences Stackedinsect resistant and herbicide tolerant LLC corn hybrid derived fromconventional cross- breeding of the parental lines 1507 (OECDidentifier: DAS-O15O7-1) and NK603 (OECD identifier: MON-OO6O3-6).TC1507 × DAS-59122-7 DOW AgroSciences Stacked insect resistant andherbicide tolerant LLC and Pioneer Hi- maize produced by conventionalcross breeding Bred International Inc. of parental lines TC1507 (OECDunique identifier: DAS-O15O7-1) with DAS-59122-7 (OECD uniqueidentifier: DAS-59122-7). Resistance to Lepidopteran insects is derivedfrom TC1507 due the presence of the Cry1F gene from Bacillusthuringiensis var. aizawai. Corn rootworm-resistance is derived fromDAS- 59122-7 which contains the Cry34Ab1 and Cry35Ab1 genes fromBacillus thuringiensis strain P5149B1. Tolerance to glufosinate ammoniumherbicide is derived from TC1507 from the phosphinothricinN-acetyltransferase encoding gene from Streptomyces viridochromogenes.

Other events with regulatory approval are well known to one skilled inthe art and can be found at the Center for Environmental Risk Assessment(cera-gmc.org/?action=gm_crop_database, which can be accessed using thewww prefix) and at the International Service for the Acquisition ofAgri-Biotech Applications (isaaa.org/gmapprovaldatabase/default.asp,which can be accessed using the www prefix).

Gene Silencing

In some embodiments the stacked trait may be in the form of silencing ofone or more polynucleotides of interest resulting in suppression of oneor more target pest polypeptides. In some embodiments the silencing isachieved through the use of a suppression DNA construct.

In some embodiments one or more polynucleotide encoding the polypeptidesof the PIP-72 polypeptides or fragments or variants thereof may bestacked with one or more polynucleotides encoding one or morepolypeptides having insecticidal activity or agronomic traits as setforth supra and optionally may further include one or morepolynucleotides providing for gene silencing of one or more targetpolynucleotides as discussed infra.

“Suppression DNA construct” is a recombinant DNA construct which whentransformed or stably integrated into the genome of the plant, resultsin “silencing” of a target gene in the plant. The target gene may beendogenous or transgenic to the plant. “Silencing,” as used herein withrespect to the target gene, refers generally to the suppression oflevels of mRNA or protein/enzyme expressed by the target gene, and/orthe level of the enzyme activity or protein functionality. The term“suppression” includes lower, reduce, decline, decrease, inhibit,eliminate and prevent. “Silencing” or “gene silencing” does not specifymechanism and is inclusive, and not limited to, anti-sense,cosuppression, viral-suppression, hairpin suppression, stem-loopsuppression, RNAi-based approaches and small RNA-based approaches.

A suppression DNA construct may comprise a region derived from a targetgene of interest and may comprise all or part of the nucleic acidsequence of the sense strand (or antisense strand) of the target gene ofinterest. Depending upon the approach to be utilized, the region may be100% identical or less than 100% identical (e.g., at least 50% or anyinteger between 51% and 100% identical) to all or part of the sensestrand (or antisense strand) of the gene of interest.

Suppression DNA constructs are well-known in the art, are readilyconstructed once the target gene of interest is selected, and include,without limitation, cosuppression constructs, antisense constructs,viral-suppression constructs, hairpin suppression constructs, stem-loopsuppression constructs, double-stranded RNA-producing constructs, andmore generally, RNAi (RNA interference) constructs and small RNAconstructs such as siRNA (short interfering RNA) constructs and miRNA(microRNA) constructs.

“Antisense inhibition” refers to the production of antisense RNAtranscripts capable of suppressing the expression of the target protein.

“Antisense RNA” refers to an RNA transcript that is complementary to allor part of a target primary transcript or mRNA and that blocks theexpression of a target isolated nucleic acid fragment (U.S. Pat. No.5,107,065). The complementarity of an antisense RNA may be with any partof the specific gene transcript, i.e., at the 5′ non-coding sequence, 3′non-coding sequence, introns or the coding sequence.

“Cosuppression” refers to the production of sense RNA transcriptscapable of suppressing the expression of the target protein. “Sense” RNArefers to RNA transcript that includes the mRNA and can be translatedinto protein within a cell or in vitro. Cosuppression constructs inplants have been previously designed by focusing on overexpression of anucleic acid sequence having homology to a native mRNA, in the senseorientation, which results in the reduction of all RNA having homologyto the overexpressed sequence (see, Vaucheret, et al., (1998) Plant J.16:651-659 and Gura, (2000) Nature 404:804-808).

Another variation describes the use of plant viral sequences to directthe suppression of proximal mRNA encoding sequences (PCT Publication WO1998/36083).

Recent work has described the use of “hairpin” structures thatincorporate all or part, of an mRNA encoding sequence in a complementaryorientation that results in a potential “stem-loop” structure for theexpressed RNA (PCT Publication WO 1999/53050). In this case the stem isformed by polynucleotides corresponding to the gene of interest insertedin either sense or anti-sense orientation with respect to the promoterand the loop is formed by some polynucleotides of the gene of interest,which do not have a complement in the construct. This increases thefrequency of cosuppression or silencing in the recovered transgenicplants. For review of hairpin suppression, see, Wesley, et al., (2003)Methods in Molecular Biology, Plant Functional Genomics: Methods andProtocols 236:273-286.

A construct where the stem is formed by at least 30 nucleotides from agene to be suppressed and the loop is formed by a random nucleotidesequence has also effectively been used for suppression (PCT PublicationWO 1999/61632).

The use of poly-T and poly-A sequences to generate the stem in thestem-loop structure has also been described (PCT Publication WO2002/00894).

Yet another variation includes using synthetic repeats to promoteformation of a stem in the stem-loop structure. Transgenic organismsprepared with such recombinant DNA fragments have been shown to havereduced levels of the protein encoded by the nucleotide fragment formingthe loop as described in PCT Publication WO 2002/00904.

RNA interference refers to the process of sequence-specificpost-transcriptional gene silencing in animals mediated by shortinterfering RNAs (siRNAs) (Fire, et al., (1998) Nature 391:806). Thecorresponding process in plants is commonly referred to aspost-transcriptional gene silencing (PTGS) or RNA silencing and is alsoreferred to as quelling in fungi. The process of post-transcriptionalgene silencing is thought to be an evolutionarily-conserved cellulardefense mechanism used to prevent the expression of foreign genes and iscommonly shared by diverse flora and phyla (Fire, et al., (1999) TrendsGenet. 15:358). Such protection from foreign gene expression may haveevolved in response to the production of double-stranded RNAs (dsRNAs)derived from viral infection or from the random integration oftransposon elements into a host genome via a cellular response thatspecifically destroys homologous single-stranded RNA of viral genomicRNA. The presence of dsRNA in cells triggers the RNAi response through amechanism that has yet to be fully characterized.

The presence of long dsRNAs in cells stimulates the activity of aribonuclease III enzyme referred to as dicer. Dicer is involved in theprocessing of the dsRNA into short pieces of dsRNA known as shortinterfering RNAs (siRNAs) (Berstein, et al., (2001) Nature 409:363).Short interfering RNAs derived from dicer activity are typically about21 to about 23 nucleotides in length and comprise about 19 base pairduplexes (Elbashir, et al., (2001) Genes Dev. 15:188). Dicer has alsobeen implicated in the excision of 21- and 22-nucleotide small temporalRNAs (stRNAs) from precursor RNA of conserved structure that areimplicated in translational control (Hutvagner, et al., (2001) Science293:834). The RNAi response also features an endonuclease complex,commonly referred to as an RNA-induced silencing complex (RISC), whichmediates cleavage of single-stranded RNA having sequence complementarityto the antisense strand of the siRNA duplex. Cleavage of the target RNAtakes place in the middle of the region complementary to the antisensestrand of the siRNA duplex (Elbashir, et al., (2001) Genes Dev. 15:188).In addition, RNA interference can also involve small RNA (e.g., miRNA)mediated gene silencing, presumably through cellular mechanisms thatregulate chromatin structure and thereby prevent transcription of targetgene sequences (see, e.g., Allshire, (2002) Science 297:1818-1819;Volpe, et al., (2002) Science 297:1833-1837; Jenuwein, (2002) Science297:2215-2218 and Hall, et al., (2002) Science 297:2232-2237). As such,miRNA molecules of the disclosure can be used to mediate gene silencingvia interaction with RNA transcripts or alternately by interaction withparticular gene sequences, wherein such interaction results in genesilencing either at the transcriptional or post-transcriptional level.

Methods and compositions are further provided which allow for anincrease in RNAi produced from the silencing element. In suchembodiments, the methods and compositions employ a first polynucleotidecomprising a silencing element for a target pest sequence operablylinked to a promoter active in the plant cell; and, a secondpolynucleotide comprising a suppressor enhancer element comprising thetarget pest sequence or an active variant or fragment thereof operablylinked to a promoter active in the plant cell. The combined expressionof the silencing element with suppressor enhancer element leads to anincreased amplification of the inhibitory RNA produced from thesilencing element over that achievable with only the expression of thesilencing element alone. In addition to the increased amplification ofthe specific RNAi species itself, the methods and compositions furtherallow for the production of a diverse population of RNAi species thatcan enhance the effectiveness of disrupting target gene expression. Assuch, when the suppressor enhancer element is expressed in a plant cellin combination with the silencing element, the methods and compositioncan allow for the systemic production of RNAi throughout the plant; theproduction of greater amounts of RNAi than would be observed with justthe silencing element construct alone; and, the improved loading of RNAiinto the phloem of the plant, thus providing better control of phloemfeeding insects by an RNAi approach. Thus, the various methods andcompositions provide improved methods for the delivery of inhibitory RNAto the target organism. See, for example, US Patent ApplicationPublication 2009/0188008.

As used herein, a “suppressor enhancer element” comprises apolynucleotide comprising the target sequence to be suppressed or anactive fragment or variant thereof. It is recognize that the suppressorenhancer element need not be identical to the target sequence, butrather, the suppressor enhancer element can comprise a variant of thetarget sequence, so long as the suppressor enhancer element hassufficient sequence identity to the target sequence to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element. Similarly,the suppressor enhancer element can comprise a fragment of the targetsequence, wherein the fragment is of sufficient length to allow for anincreased level of the RNAi produced by the silencing element over thatachievable with only the expression of the silencing element.

It is recognized that multiple suppressor enhancer elements from thesame target sequence or from different target sequences or fromdifferent regions of the same target sequence can be employed. Forexample, the suppressor enhancer elements employed can comprisefragments of the target sequence derived from different region of thetarget sequence (i.e., from the 3′UTR, coding sequence, intron, and/or5′UTR). Further, the suppressor enhancer element can be contained in anexpression cassette, as described elsewhere herein, and in specificembodiments, the suppressor enhancer element is on the same or on adifferent DNA vector or construct as the silencing element. Thesuppressor enhancer element can be operably linked to a promoter asdisclosed herein. It is recognized that the suppressor enhancer elementcan be expressed constitutively or alternatively, it may be produced ina stage-specific manner employing the various inducible ortissue-preferred or developmentally regulated promoters that arediscussed elsewhere herein.

In specific embodiments, employing both a silencing element and thesuppressor enhancer element the systemic production of RNAi occursthroughout the entire plant. In further embodiments, the plant or plantparts of the disclosure have an improved loading of RNAi into the phloemof the plant than would be observed with the expression of the silencingelement construct alone and, thus provide better control of phloemfeeding insects by an RNAi approach. In specific embodiments, theplants, plant parts and plant cells of the disclosure can further becharacterized as allowing for the production of a diversity of RNAispecies that can enhance the effectiveness of disrupting target geneexpression.

In specific embodiments, the combined expression of the silencingelement and the suppressor enhancer element increases the concentrationof the inhibitory RNA in the plant cell, plant, plant part, plant tissueor phloem over the level that is achieved when the silencing element isexpressed alone.

As used herein, an “increased level of inhibitory RNA” comprises anystatistically significant increase in the level of RNAi produced in aplant having the combined expression when compared to an appropriatecontrol plant. For example, an increase in the level of RNAi in theplant, plant part or the plant cell can comprise at least about a 1%,about a 1%-5%, about a 5%-10%, about a 10%-20%, about a 20%-30%, about a30%-40%, about a 40%-50%, about a 50%-60%, about 60-70%, about 70%-80%,about a 80%-90%, about a 90%-100% or greater increase in the level ofRNAi in the plant, plant part, plant cell or phloem when compared to anappropriate control. In other embodiments, the increase in the level ofRNAi in the plant, plant part, plant cell or phloem can comprise atleast about a 1 fold, about a 1 fold-5 fold, about a 5 fold-10 fold,about a 10 fold-20 fold, about a 20 fold-30 fold, about a 30 fold-40fold, about a 40 fold-50 fold, about a 50 fold-60 fold, about 60 fold-70fold, about 70 fold-80 fold, about a 80 fold-90 fold, about a 90fold-100 fold or greater increase in the level of RNAi in the plant,plant part, plant cell or phloem when compared to an appropriatecontrol. Examples of combined expression of the silencing element withsuppressor enhancer element for the control of Stinkbugs and Lygus canbe found in US Patent Application Publication 2011/0301223 and US PatentApplication Publication 2009/0192117.

Some embodiments relate to down-regulation of expression of target genesin insect pest species by interfering ribonucleic acid (RNA) molecules.PCT Publication WO 2007/074405 describes methods of inhibitingexpression of target genes in invertebrate pests including Coloradopotato beetle. PCT Publication WO 2005/110068 describes methods ofinhibiting expression of target genes in invertebrate pests including inparticular Western corn rootworm as a means to control insectinfestation. Furthermore, PCT Publication WO 2009/091864 describescompositions and methods for the suppression of target genes from insectpest species including pests from the Lygus genus. Nucleic acidmolecules including RNAi for targeting the vacuolar ATPase H subunit,useful for controlling a coleopteran pest population and infestation asdescribed in US Patent Application Publication 2012/0198586. PCTPublication WO 2012/055982 describes ribonucleic acid (RNA or doublestranded RNA) that inhibits or down regulates the expression of a targetgene that encodes: an insect ribosomal protein such as the ribosomalprotein L19, the ribosomal protein L40 or the ribosomal protein S27A; aninsect proteasome subunit such as the Rpn6 protein, the Pros 25, theRpn2 protein, the proteasome beta 1 subunit protein or the Pros beta 2protein; an insect β-coatomer of the COPI vesicle, the γ-coatomer of theCOPI vesicle, the β′-coatomer protein or the ζ-coatomer of the COPIvesicle; an insect Tetraspanine 2 A protein which is a putativetransmembrane domain protein; an insect protein belonging to the actinfamily such as Actin 5C; an insect ubiquitin-5E protein; an insect Sec23protein which is a GTPase activator involved in intracellular proteintransport; an insect crinkled protein which is an unconventional myosinwhich is involved in motor activity; an insect crooked neck proteinwhich is involved in the regulation of nuclear alternative mRNAsplicing; an insect vacuolar H+-ATPase G-subunit protein and an insectTbp-1 such as Tat-binding protein. US Patent Application Publications2012/029750, US 20120297501, and 2012/0322660 describe interferingribonucleic acids (RNA or double stranded RNA) that functions uponuptake by an insect pest species to down-regulate expression of a targetgene in said insect pest, wherein the RNA comprises at least onesilencing element wherein the silencing element is a region ofdouble-stranded RNA comprising annealed complementary strands, onestrand of which comprises or consists of a sequence of nucleotides whichis at least partially complementary to a target nucleotide sequencewithin the target gene. US Patent Application Publication 2012/0164205describes potential targets for interfering double stranded ribonucleicacids for inhibiting invertebrate pests including: a Chd3 HomologousSequence, a Beta-Tubulin Homologous Sequence, a 40 kDa V-ATPaseHomologous Sequence, a EF1α Homologous Sequence, a 26S ProteosomeSubunit p28 Homologous Sequence, a Juvenile Hormone Epoxide HydrolaseHomologous Sequence, a Swelling Dependent Chloride Channel ProteinHomologous Sequence, a Glucose-6-Phosphate 1-Dehydrogenase ProteinHomologous Sequence, an Act42A Protein Homologous Sequence, aADP-Ribosylation Factor 1 Homologous Sequence, a Transcription FactorIIB Protein Homologous Sequence, a Chitinase Homologous Sequences, aUbiquitin Conjugating Enzyme Homologous Sequence, aGlyceraldehyde-3-Phosphate Dehydrogenase Homologous Sequence, anUbiquitin B Homologous Sequence, a Juvenile Hormone Esterase Homolog,and an Alpha Tubuliln Homologous Sequence. US Patent ApplicationPublication 2009/0192117 describes suppression of target polynucleotidesfrom Lygus.

Use in Pesticidal Control

General methods for employing strains comprising a nucleic acid sequenceof the embodiments or a variant thereof, in pesticide control or inengineering other organisms as pesticidal agents are known in the art.See, for example U.S. Pat. No. 5,039,523 and EP 0480762A2.

Microorganism hosts that are known to occupy the “phytosphere”(phylloplane, phyllosphere, rhizosphere, and/or rhizoplana) of one ormore crops of interest may be selected. These microorganisms areselected so as to be capable of successfully competing in the particularenvironment with the wild-type microorganisms, provide for stablemaintenance and expression of the gene expressing the PIP-72polypeptide, and desirably, provide for improved protection of thepesticide from environmental degradation and inactivation.

Such microorganisms include bacteria, algae, and fungi. Of particularinterest are microorganisms such as bacteria, e.g., Pseudomonas,Erwinia, Serratia, Klebsiella, Xanthomonas, Streptomyces, Rhizobium,Rhodopseudomonas, Methylius, Agrobacterium, Acetobacter, Lactobacillus,Arthrobacter, Azotobacter, Leuconostoc, and Alcaligenes, fungi,particularly yeast, e.g., Saccharomyces, Cryptococcus, Kluyveromyces,Sporobolomyces, Rhodotorula, and Aureobasidium. Of particular interestare such phytosphere bacterial species as Pseudomonas syringae,Pseudomonas fluorescens, Pseudomonas chlororaphis, Serratia marcescens,Acetobacter xylinum, Agrobacteria, Rhodopseudomonas spheroides,Xanthomonas campestris, Rhizobium melioti, Alcaligenes entrophus,Clavibacter xyli and Azotobacter vinelandii and phytosphere yeastspecies such as Rhodotorula rubra, R. glutinis, R. marina, R.aurantiaca, Cryptococcus albidus, C. diffluens, C. laurentii,Saccharomyces rosei, S. pretoriensis, S. cerevisiae, Sporobolomycesroseus, S. odorus, Kluyveromyces veronae, and Aureobasidium pollulans.Of particular interest are the pigmented microorganisms. Host organismsof particular interest include yeast, such as Rhodotorula spp.,Aureobasidium spp., Saccharomyces spp. (such as S. cerevisiae),Sporobolomyces spp., phylloplane organisms such as Pseudomonas spp.(such as P. aeruginosa, P. fluorescens, P. chlororaphis), Erwinia spp.,and Flavobacterium spp., and other such organisms, includingAgrobacterium tumefaciens, E. coli, Bacillus subtilis, Bacillus cereusand the like.

Genes encoding the PIP-72 polypeptides of the embodiments can beintroduced into microorganisms that multiply on plants (epiphytes) todeliver PIP-72 polypeptides to potential target pests. Epiphytes, forexample, can be gram-positive or gram-negative bacteria.

Root-colonizing bacteria, for example, can be isolated from the plant ofinterest by methods known in the art. Specifically, a Bacillus cereusstrain that colonizes roots can be isolated from roots of a plant (see,for example, Handelsman et al. (1991) Appl. Environ. Microbiol.56:713-718). Genes encoding the PIP-72 polypeptides of the embodimentscan be introduced into a root-colonizing Bacillus cereus by standardmethods known in the art.

Genes encoding PIP-72 polypeptides can be introduced, for example, intothe root-colonizing Bacillus by means of electro transformation.Specifically, genes encoding the PIP-72 polypeptides can be cloned intoa shuttle vector, for example, pHT3101 (Lerecius, et al., (1989) FEMSMicrobiol. Letts. 60:211-218. The shuttle vector pHT3101 containing thecoding sequence for the particular PIP-72 polypeptide gene can, forexample, be transformed into the root-colonizing Bacillus by means ofelectroporation (Lerecius, et al., (1989) FEMS Microbiol. Letts.60:211-218).

Expression systems can be designed so that PIP-72 polypeptides aresecreted outside the cytoplasm of gram-negative bacteria, such as E.coli, for example. Advantages of having PIP-72 polypeptides secretedare: (1) avoidance of potential cytotoxic effects of the PIP-72polypeptide expressed; and (2) improvement in the efficiency ofpurification of the PIP-72 polypeptide, including, but not limited to,increased efficiency in the recovery and purification of the protein pervolume cell broth and decreased time and/or costs of recovery andpurification per unit protein.

PIP-72 polypeptides can be made to be secreted in E. coli, for example,by fusing an appropriate E. coli signal peptide to the amino-terminalend of the PIP-72 polypeptide. Signal peptides recognized by E. coli canbe found in proteins already known to be secreted in E. coli, forexample the OmpA protein (Ghrayeb, et al., (1984) EMBO J, 3:2437-2442).OmpA is a major protein of the E. coli outer membrane, and thus itssignal peptide is thought to be efficient in the translocation process.Also, the OmpA signal peptide does not need to be modified beforeprocessing as may be the case for other signal peptides, for examplelipoprotein signal peptide (Duffaud, et al., (1987) Meth. Enzymol.153:492).

PIP-72 polypeptides of the embodiments can be fermented in a bacterialhost and the resulting bacteria processed and used as a microbial sprayin the same manner that Bt strains have been used as insecticidalsprays. In the case of a PIP-72 polypeptide(s) that is secreted fromBacillus, the secretion signal is removed or mutated using proceduresknown in the art. Such mutations and/or deletions prevent secretion ofthe PIP-72 polypeptide(s) into the growth medium during the fermentationprocess. The PIP-72 polypeptides are retained within the cell, and thecells are then processed to yield the encapsulated PIP-72 polypeptides.Any suitable microorganism can be used for this purpose. Pseudomonas hasbeen used to express Bt toxins as encapsulated proteins and theresulting cells processed and sprayed as an insecticide (Gaertner, etal., (1993), in: Advanced Engineered Pesticides, ed. Kim).

Alternatively, the PIP-72 polypeptides are produced by introducing aheterologous gene into a cellular host. Expression of the heterologousgene results, directly or indirectly, in the intracellular productionand maintenance of the pesticide. These cells are then treated underconditions that prolong the activity of the toxin produced in the cellwhen the cell is applied to the environment of target pest(s). Theresulting product retains the toxicity of the toxin. These naturallyencapsulated PIP-72 polypeptides may then be formulated in accordancewith conventional techniques for application to the environment hostinga target pest, e.g., soil, water, and foliage of plants. See, forexample EPA 0192319, and the references cited therein.

Pesticidal Compositions

In some embodiments the active ingredients can be applied in the form ofcompositions and can be applied to the crop area or plant to be treated,simultaneously or in succession, with other compounds. These compoundscan be fertilizers, weed killers, Cryoprotectants, surfactants,detergents, pesticidal soaps, dormant oils, polymers, and/ortime-release or biodegradable carrier formulations that permit long-termdosing of a target area following a single application of theformulation. They can also be selective herbicides, chemicalinsecticides, virucides, microbicides, amoebicides, pesticides,fungicides, bacteriocides, nematocides, molluscicides or mixtures ofseveral of these preparations, if desired, together with furtheragriculturally acceptable carriers, surfactants or application-promotingadjuvants customarily employed in the art of formulation. Suitablecarriers and adjuvants can be solid or liquid and correspond to thesubstances ordinarily employed in formulation technology, e.g. naturalor regenerated mineral substances, solvents, dispersants, wettingagents, tackifiers, binders or fertilizers. Likewise the formulationsmay be prepared into edible “baits” or fashioned into pest “traps” topermit feeding or ingestion by a target pest of the pesticidalformulation.

Methods of applying an active ingredient or an agrochemical compositionthat contains at least one of the PIP-72 polypeptides produced by thebacterial strains include leaf application, seed coating and soilapplication. The number of applications and the rate of applicationdepend on the intensity of infestation by the corresponding pest.

The composition may be formulated as a powder, dust, pellet, granule,spray, emulsion, colloid, solution or such like, and may be prepared bysuch conventional means as desiccation, lyophilization, homogenation,extraction, filtration, centrifugation, sedimentation or concentrationof a culture of cells comprising the polypeptide. In all suchcompositions that contain at least one such pesticidal polypeptide, thepolypeptide may be present in a concentration of from about 1% to about99% by weight.

Lepidopteran, Dipteran, Heteropteran, nematode, Hemiptera or Coleopteranpests may be killed or reduced in numbers in a given area by the methodsof the disclosure or may be prophylactically applied to an environmentalarea to prevent infestation by a susceptible pest. Preferably the pestingests or is contacted with, a pesticidally-effective amount of thepolypeptide. “Pesticidally-effective amount” as used herein refers to anamount of the pesticide that is able to bring about death to at leastone pest or to noticeably reduce pest growth, feeding or normalphysiological development. This amount will vary depending on suchfactors as, for example, the specific target pests to be controlled, thespecific environment, location, plant, crop or agricultural site to betreated, the environmental conditions and the method, rate,concentration, stability, and quantity of application of thepesticidally-effective polypeptide composition. The formulations mayalso vary with respect to climatic conditions, environmentalconsiderations, and/or frequency of application and/or severity of pestinfestation.

The pesticide compositions described may be made by formulating eitherthe bacterial cell, Crystal and/or spore suspension or isolated proteincomponent with the desired agriculturally-acceptable carrier. Thecompositions may be formulated prior to administration in an appropriatemeans such as lyophilized, freeze-dried, desiccated or in an aqueouscarrier, medium or suitable diluent, such as saline or other buffer. Theformulated compositions may be in the form of a dust or granularmaterial or a suspension in oil (vegetable or mineral) or water oroil/water emulsions or as a wettable powder or in combination with anyother carrier material suitable for agricultural application. Suitableagricultural carriers can be solid or liquid and are well known in theart. The term “agriculturally-acceptable carrier” covers all adjuvants,inert components, dispersants, surfactants, tackifiers, binders, etc.that are ordinarily used in pesticide formulation technology; these arewell known to those skilled in pesticide formulation. The formulationsmay be mixed with one or more solid or liquid adjuvants and prepared byvarious means, e.g., by homogeneously mixing, blending and/or grindingthe pesticidal composition with suitable adjuvants using conventionalformulation techniques. Suitable formulations and application methodsare described in U.S. Pat. No. 6,468,523, herein incorporated byreference. The plants can also be treated with one or more chemicalcompositions, including one or more herbicide, insecticides orfungicides.

Exemplary chemical compositions include: Fruits/Veqetables Herbicides:Atrazine, Bromacil, Diuron, Glyphosate, Linuron, Metribuzin, Simazine,Trifluralin, Fluazifop, Glufosinate, Halo sulfuron Gowan, Paraquat,Propyzamide, Sethoxydim, Butafenacil, Halosulfuron, Indaziflam;Fruits/Veqetables Insecticides: Aldicarb, Bacillus thuriengiensis,Carbaryl, Carbofuran, Chlorpyrifos, Cypermethrin, Deltamethrin,Diazinon, Malathion, Abamectin, Cyfluthrin/beta-cyfluthrin,Esfenvalerate, Lambda-cyhalothrin, Acequinocyl, Bifenazate,Methoxyfenozide, Novaluron, Chromafenozide, Thiacloprid, Dinotefuran,FluaCrypyrim, Tolfenpyrad, Clothianidin, Spirodiclofen,Gamma-cyhalothrin, Spiromesifen, Spinosad, Rynaxypyr, Cyazypyr,Spinoteram, Triflumuron, Spirotetramat, Imidacloprid, Flubendiamide,Thiodicarb, Metaflumizone, Sulfoxaflor, Cyflumetofen, Cyanopyrafen,Imidacloprid, Clothianidin, Thiamethoxam, Spinotoram, Thiodicarb,Flonicamid, Methiocarb, Emamectin-benzoate, Indoxacarb, Forthiazate,Fenamiphos, Cadusaphos, Pyriproxifen, Fenbutatin-oxid, Hexthiazox,Methomyl,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on;Fruits/Vegetables Fungicides: Carbendazim, Chlorothalonil, EBDCs,Sulphur, Thiophanate-methyl, Azoxystrobin, Cymoxanil, Fluazinam,Fosetyl, Iprodione, Kresoxim-methyl, Metalaxyl/mefenoxam,Trifloxystrobin, Ethaboxam, Iprovalicarb, Trifloxystrobin, Fenhexamid,Oxpoconazole fumarate, Cyazofamid, Fenamidone, Zoxamide, Picoxystrobin,Pyraclostrobin, Cyflufenamid, Boscalid; Cereals Herbicides: Isoproturon,Bromoxynil, loxynil, Phenoxies, Chlorsulfuron, Clodinafop, Diclofop,Diflufenican, Fenoxaprop, Florasulam, Fluoroxypyr, Metsulfuron,Triasulfuron, Flucarbazone, lodosulfuron, Propoxycarbazone, Picolinafen,Mesosulfuron, Beflubutamid, Pinoxaden, Amidosulfuron, ThifensulfuronMethyl, Tribenuron, Flupyrsulfuron, Sulfosulfuron, Pyrasulfotole,Pyroxsulam, Flufenacet, Tralkoxydim, Pyroxasulfon; Cereals Fungicides:Carbendazim, Chlorothalonil, Azoxystrobin, Cyproconazole, Cyprodinil,Fenpropimorph, Epoxiconazole, Kresoxim-methyl, Quinoxyfen, Tebuconazole,Trifloxystrobin, Simeconazole, Picoxystrobin, Pyraclostrobin,Dimoxystrobin, Prothioconazole, Fluoxastrobin; Cereals Insecticides:Dimethoate, Lambda-cyhalthrin, Deltamethrin, alpha-Cypermethrin,β-cyfluthrin, Bifenthrin, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Clorphyriphos, Metamidophos,Oxidemethon-methyl, Pirimicarb, Methiocarb; Maize Herbicides: Atrazine,Alachlor, Bromoxynil, Acetochlor, Dicamba, Clopyralid, (S-)Dimethenamid, Glufosinate, Glyphosate, Isoxaflutole, (S-)Metolachlor,Mesotrione, Nicosulfuron, Primisulfuron, Rimsulfuron, Sulcotrione,Foramsulfuron, Topramezone, Tembotrione, Saflufenacil, Thiencarbazone,Flufenacet, Pyroxasulfon; Maize Insecticides: Carbofuran, Chlorpyrifos,Bifenthrin, Fipronil, Imidacloprid, Lambda-Cyhalothrin, Tefluthrin,Terbufos, Thiamethoxam, Clothianidin, Spiromesifen, Flubendiamide,Triflumuron, Rynaxypyr, Deltamethrin, Thiodicarb, β-Cyfluthrin,Cypermethrin, Bifenthrin, Lufenuron, Triflumoron, Tefluthrin,Tebupirimphos, Ethiprole, Cyazypyr, Thiacloprid, Acetamiprid,Dinetofuran, Avermectin, Methiocarb, Spirodiclofen, Spirotetramat; MaizeFungicides: Fenitropan, Thiram, Prothioconazole, Tebuconazole,Trifloxystrobin; Rice Herbicides: Butachlor, Propanil, Azimsulfuron,Bensulfuron, Cyhalofop, Daimuron, Fentrazamide, Imazosulfuron,Mefenacet, Oxaziclomefone, Pyrazosulfuron, Pyributicarb, Quinclorac,Thiobencarb, Indanofan, Flufenacet, Fentrazamide, Halosulfuron,Oxaziclomefone, Benzobicyclon, Pyriftalid, Penoxsulam, Bispyribac,Oxadiargyl, Ethoxysulfuron, Pretilachlor, Mesotrione, Tefuryltrione,Oxadiazone, Fenoxaprop, Pyrimisulfan; Rice Insecticides: Diazinon,Fenitrothion, Fenobucarb, Monocrotophos, Benfuracarb, Buprofezin,Dinotefuran, Fipronil, Imidacloprid, Isoprocarb, Thiacloprid,Chromafenozide, Thiacloprid, Dinotefuran, Clothianidin, Ethiprole,Flubendiamide, Rynaxypyr, Deltamethrin, Acetamiprid, Thiamethoxam,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Cypermethrin,Chlorpyriphos, Cartap, Methamidophos, Etofenprox, Triazophos,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Carbofuran, Benfuracarb; Rice Funaicides: Thiophanate-methyl,Azoxystrobin, Carpropamid, Edifenphos, Ferimzone, Iprobenfos,Isoprothiolane, Pencycuron, Probenazole, Pyroquilon, Tricyclazole,Trifloxystrobin, Diclocymet, Fenoxanil, Simeconazole, Tiadinil; CottonHerbicides: Diuron, Fluometuron, MSMA, Oxyfluorfen, Prometryn,Trifluralin, Carfentrazone, Clethodim, Fluazifop-butyl, Glyphosate,Norflurazon, Pendimethalin, Pyrithiobac-sodium, Trifloxysulfuron,Tepraloxydim, Glufosinate, Flumioxazin, Thidiazuron; CottonInsecticides: Acephate, Aldicarb, Chlorpyrifos, Cypermethrin,Deltamethrin, Malathion, Monocrotophos, Abamectin, Acetamiprid,Emamectin Benzoate, Imidacloprid, Indoxacarb, Lambda-Cyhalothrin,Spinosad, Thiodicarb, Gamma-Cyhalothrin, Spiromesifen, Pyridalyl,Flonicamid, Flubendiamide, Triflumuron, Rynaxypyr, Beta-Cyfluthrin,Spirotetramat, Clothianidin, Thiamethoxam, Thiacloprid, Dinetofuran,Flubendiamide, Cyazypyr, Spinosad, Spinotoram, gamma Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Thiodicarb, Avermectin, Flonicamid, Pyridalyl, Spiromesifen,Sulfoxaflor, Profenophos, Thriazophos, Endosulfan; Cotton Fungicides:Etridiazole, Metalaxyl, Quintozene; Soybean Herbicides: Alachlor,Bentazone, Trifluralin, Chlorimuron-Ethyl, Cloransulam-Methyl,Fenoxaprop, Fomesafen, Fluazifop, Glyphosate, Imazamox, Imazaquin,Imazethapyr, (S-)Metolachlor, Metribuzin, Pendimethalin, Tepraloxydim,Glufosinate; Soybean Insecticides: Lambda-cyhalothrin, Methomyl,Parathion, Thiocarb, Imidacloprid, Clothianidin, Thiamethoxam,Thiacloprid, Acetamiprid, Dinetofuran, Flubendiamide, Rynaxypyr,Cyazypyr, Spinosad, Spinotoram, Emamectin-Benzoate, Fipronil, Ethiprole,Deltamethrin, β-Cyfluthrin, gamma and lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Spirotetramat, Spinodiclofen, Triflumuron, Flonicamid, Thiodicarb,beta-Cyfluthrin; Soybean Fungicides: Azoxystrobin, Cyproconazole,Epoxiconazole, Flutriafol, Pyraclostrobin, Tebuconazole,Trifloxystrobin, Prothioconazole, Tetraconazole; Sugarbeet Herbicides:Chloridazon, Desmedipham, Ethofumesate, Phenmedipham, Triallate,Clopyralid, Fluazifop, Lenacil, Metamitron, Quinmerac, Cycloxydim,Triflusulfuron, Tepraloxydim, Quizalofop; Sugarbeet Insecticides:Imidacloprid, Clothianidin, Thiamethoxam, Thiacloprid, Acetamiprid,Dinetofuran, Deltamethrin, β-Cyfluthrin, gamma/lambda Cyhalothrin,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on,Tefluthrin, Rynaxypyr, Cyaxypyr, Fipronil, Carbofuran; CanolaHerbicides: Clopyralid, Diclofop, Fluazifop, Glufosinate, Glyphosate,Metazachlor, Trifluralin Ethametsulfuron, Quinmerac, Quizalofop,Clethodim, Tepraloxydim; Canola Fungicides: Azoxystrobin, Carbendazim,Fludioxonil, Iprodione, Prochloraz, Vinclozolin; Canola Insecticides:Carbofuran organophosphates, Pyrethroids, Thiacloprid, Deltamethrin,Imidacloprid, Clothianidin, Thiamethoxam, Acetamiprid, Dinetofuran,β-Cyfluthrin, gamma and lambda Cyhalothrin, tau-Fluvaleriate, Ethiprole,Spinosad, Spinotoram, Flubendiamide, Rynaxypyr, Cyazypyr,4-[[(6-Chlorpyridin-3-yl)methyl](2,2-difluorethyl)amino]furan-2(5H)-on.

In some embodiments the herbicide is Atrazine, Bromacil, Diuron,Chlorsulfuron, Metsulfuron, Thifensulfuron Methyl, Tribenuron,Acetochlor, Dicamba, Isoxaflutole, Nicosulfuron, Rimsulfuron,Pyrithiobac-sodium, Flumioxazin, Chlorimuron-Ethyl, Metribuzin,Quizalofop, S-metolachlor, Hexazinne or combinations thereof.

In some embodiments the insecticide is Esfenvalerate,Chlorantraniliprole, Methomyl, Indoxacarb, Oxamyl or combinationsthereof.

In some embodiment the herbicide is a homogentisate solanesyltransferase(HST) inhibiting herbicide and/or hydroxyphenyl pyruvate dioxygenase(HPPD) inhibiting herbicide of US Patent Publication US2011173718.

Pesticidal and Insecticidal Activity

“Pest” includes but is not limited to, insects, fungi, bacteria,nematodes, mites, ticks and the like. Insect pests include insectsselected from the orders Coleoptera, Diptera, Hymenoptera, Lepidoptera,Mallophaga, Homoptera, Hemiptera Orthroptera, Thysanoptera, Dermaptera,Isoptera, Anoplura, Siphonaptera, Trichoptera, etc., particularlyLepidoptera and Coleoptera.

Those skilled in the art will recognize that not all compounds areequally effective against all pests. Compounds of the embodimentsdisplay activity against insect pests, which may include economicallyimportant agronomic, forest, greenhouse, nursery ornamentals, food andfiber, public and animal health, domestic and commercial structure,household and stored product pests.

Larvae of the order Lepidoptera include, but are not limited to,armyworms, cutworms, loopers and heliothines in the family NoctuidaeSpodoptera frugiperda J E Smith (fall armyworm); S. exigua Hübner (beetarmyworm); S. litura Fabricius (tobacco cutworm, cluster caterpillar);Mamestra configurata Walker (bertha armyworm); M. brassicae Linnaeus(cabbage moth); Agrotis ipsilon Hufnagel (black cutworm); A. orthogoniaMorrison (western cutworm); A. subterranea Fabricius (granulatecutworm); Alabama argillacea Hübner (cotton leaf worm); Trichoplusia niHübner (cabbage looper); Pseudoplusia includens Walker (soybean looper);Anticarsia gemmatalis Hübner (velvetbean caterpillar); Hypena scabraFabricius (green cloverworm); Heliothis virescens Fabricius (tobaccobudworm); Pseudaletia unipuncta Haworth (armyworm); Athetis mindaraBarnes and Mcdunnough (rough skinned cutworm); Euxoa messoria Harris(darksided cutworm); Earias insulana Boisduval (spiny bollworm); E.vittella Fabricius (spotted bollworm); Helicoverpa armigera Hübner(American bollworm); H. zea Boddie (corn earworm or cotton bollworm);Melanchra picta Harris (zebra caterpillar); Egira (Xylomyges) curialisGrote (citrus cutworm); borers, casebearers, webworms, coneworms, andskeletonizers from the family Pyralidae Ostrinia nubilalis Hübner(European corn borer); Amyelois transitella Walker (naval orangeworm);Anagasta kuehniella Zeller (Mediterranean flour moth); Cadra cautellaWalker (almond moth); Chilo suppressalis Walker (rice stem borer); C.partellus, (sorghum borer); Corcyra cephalonica Stainton (rice moth);Crambus caliginosellus Clemens (corn root webworm); C. teterrellusZincken (bluegrass webworm); Cnaphalocrocis medinalis Guenée (rice leafroller); Desmia funeralis Hübner (grape leaffolder); Diaphania hyalinataLinnaeus (melon worm); D. nitidalis Stoll (pickleworm); Diatraeagrandiosella Dyar (southwestern corn borer), D. saccharalis Fabricius(surgarcane borer); Eoreuma loftini Dyar (Mexican rice borer); Ephestiaelutella Hübner (tobacco (cacao) moth); Galleria mellonella Linnaeus(greater wax moth); Herpetogramma licarsisalis Walker (sod webworm);Homoeosoma electellum Hulst (sunflower moth); Elasmopalpus lignosellusZeller (lesser cornstalk borer); Achroia grisella Fabricius (lesser waxmoth); Loxostege sticticalis Linnaeus (beet webworm); Orthaga thyrisalisWalker (tea tree web moth); Maruca testulalis Geyer (bean pod borer);Plodia interpunctella Hübner (Indian meal moth); Scirpophaga incertulasWalker (yellow stem borer); Udea rubigalis Guenée (celery leaftier); andleafrollers, budworms, seed worms and fruit worms in the familyTortricidae Acleris gloverana Walsingham (Western blackheaded budworm);A. variana Fernald (Eastern blackheaded budworm); Archips argyrospilaWalker (fruit tree leaf roller); A. rosana Linnaeus (European leafroller); and other Archips species, Adoxophyes orana Fischer vonRösslerstamm (summer fruit tortrix moth); Cochylis hospes Walsingham(banded sunflower moth); Cydia latiferreana Walsingham (filbertworm); C.pomonella Linnaeus (coding moth); Platynota flavedana Clemens(variegated leafroller); P. stultana Walsingham (omnivorous leafroller);Lobesia botrana Denis & Schiffermüller (European grape vine moth);Spilonota ocellana Denis & Schiffermüller (eyespotted bud moth);Endopiza viteana Clemens (grape berry moth); Eupoecilia ambiguellaHübner (vine moth); Bonagota salubricola Meyrick (Brazilian appleleafroller); Grapholita molesta Busck (oriental fruit moth); Suleimahelianthana Riley (sunflower bud moth); Argyrotaenia spp.; Choristoneuraspp.

Selected other agronomic pests in the order Lepidoptera include, but arenot limited to, Alsophila pometaria Harris (fall cankerworm); Anarsialineatella Zeller (peach twig borer); Anisota senatoria J. E. Smith(orange striped oakworm); Antheraea pernyi Guérin-Méneville (Chinese OakTussah Moth); Bombyx mori Linnaeus (Silkworm); Bucculatrix thurberiellaBusck (cotton leaf perforator); Colias eurytheme Boisduval (alfalfacaterpillar); Datana integerrima Grote & Robinson (walnut caterpillar);Dendrolimus sibiricus Tschetwerikov (Siberian silk moth), Ennomossubsignaria Hübner (elm spanworm); Erannis tiliaria Harris (lindenlooper); Euproctis chrysorrhoea Linnaeus (browntail moth); Harrisinaamericana Guérin-Méneville (grapeleaf skeletonizer); Hemileuca oliviaeCockrell (range caterpillar); Hyphantria cunea Drury (fall webworm);Keiferia lycopersicella Walsingham (tomato pinworm); Lambdinafiscellaria fiscellaria Hulst (Eastern hemlock looper); L. fiscellarialugubrosa Hulst (Western hemlock looper); Leucoma salicis Linnaeus(satin moth); Lymantria dispar Linnaeus (gypsy moth); Manducaquinquemaculata Haworth (five spotted hawk moth, tomato hornworm); M.sexta Haworth (tomato hornworm, tobacco hornworm); Operophtera brumataLinnaeus (winter moth); Paleacrita vernata Peck (spring cankerworm);Papilio cresphontes Cramer (giant swallowtail orange dog); Phryganidiacalifornica Packard (California oakworm); Phyllocnistis citrellaStainton (citrus leafminer); Phyllonorycter blancardella Fabricius(spotted tentiform leafminer); Pieris brassicae Linnaeus (large whitebutterfly); P. rapae Linnaeus (small white butterfly); P. napi Linnaeus(green veined white butterfly); Platyptilia carduidactyla Riley(artichoke plume moth); Plutella xylostella Linnaeus (diamondback moth);Pectinophora gossypiella Saunders (pink bollworm); Pontia protodiceBoisduval and Leconte (Southern cabbageworm); Sabulodes aegrotata Guenée(omnivorous looper); Schizura concinna J. E. Smith (red humpedcaterpillar); Sitotroga cerealella Olivier (Angoumois grain moth);Thaumetopoea pityocampa Schiffermuller (pine processionary caterpillar);Tineola bisselliella Hummel (webbing clothesmoth); Tuta absoluta Meyrick(tomato leafminer); Yponomeuta padella Linnaeus (ermine moth); Heliothissubflexa Guenée; Malacosoma spp. and Orgyia spp.

Of interest are larvae and adults of the order Coleoptera includingweevils from the families Anthribidae, Bruchidae and Curculionidae(including, but not limited to: Anthonomus grandis Boheman (bollweevil); Lissorhoptrus oryzophilus Kuschel (rice water weevil);Sitophilus granarius Linnaeus (granary weevil); S. oryzae Linnaeus (riceweevil); Hypera punctata Fabricius (clover leaf weevil);Cylindrocopturus adspersus LeConte (sunflower stem weevil); Smicronyxfulvus LeConte (red sunflower seed weevil); S. sordidus LeConte (graysunflower seed weevil); Sphenophorus maidis Chittenden (maize billbug));flea beetles, cucumber beetles, rootworms, leaf beetles, potato beetlesand leafminers in the family Chrysomelidae (including, but not limitedto: Leptinotarsa decemlineata Say (Colorado potato beetle); Diabroticavirgifera virgifera LeConte (western corn rootworm); D. barberi Smithand Lawrence (northern corn rootworm); D. undecimpunctata howardi Barber(southern corn rootworm); Chaetocnema pulicaria Melsheimer (corn fleabeetle); Phyllotreta cruciferae Goeze (Crucifer flea beetle);Phyllotreta striolata (stripped flea beetle); Colaspis brunnea Fabricius(grape colaspis); Oulema melanopus Linnaeus (cereal leaf beetle);Zygogramma exclamationis Fabricius (sunflower beetle)); beetles from thefamily Coccinellidae (including, but not limited to: Epilachnavarivestis Mulsant (Mexican bean beetle)); chafers and other beetlesfrom the family Scarabaeidae (including, but not limited to: Popilliajaponica Newman (Japanese beetle); Cyclocephala borealis Arrow (northernmasked chafer, white grub); C. immaculata Olivier (southern maskedchafer, white grub); Rhizotrogus majalis Razoumowsky (European chafer);Phyllophaga crinita Burmeister (white grub); Ligyrus gibbosus De Geer(carrot beetle)); carpet beetles from the family Dermestidae; wirewormsfrom the family Elateridae, Eleodes spp., Melanotus spp.; Conoderusspp.; Limonius spp.; Agriotes spp.; Ctenicera spp.; Aeolus spp.; barkbeetles from the family Scolytidae and beetles from the familyTenebrionidae.

Adults and immatures of the order Diptera are of interest, includingleafminers Agromyza parvicornis Loew (corn blotch leafminer); midges(including, but not limited to: Contarinia sorghicola Coquillett(sorghum midge); Mayetiola destructor Say (Hessian fly); Sitodiplosismosellana Géhin (wheat midge); Neolasioptera murtfeldtiana Felt,(sunflower seed midge)); fruit flies (Tephritidae), Oscinella fritLinnaeus (fruit flies); maggots (including, but not limited to: Deliaplatura Meigen (seedcorn maggot); D. coarctata Fallen (wheat bulb fly)and other Delia spp., Meromyza americana Fitch (wheat stem maggot);Musca domestica Linnaeus (house flies); Fannia canicularis Linnaeus, F.femoralis Stein (lesser house flies); Stomoxys calcitrans Linnaeus(stable flies)); face flies, horn flies, blow flies, Chrysomya spp.;Phormia spp. and other muscoid fly pests, horse flies Tabanus spp.; botflies Gastrophilus spp.; Oestrus spp.; cattle grubs Hypoderma spp.; deerflies Chrysops spp.; Melophagus ovinus Linnaeus (keds) and otherBrachycera, mosquitoes Aedes spp.; Anopheles spp.; Culex spp.; blackflies Prosimulium spp.; Simulium spp.; biting midges, sand flies,sciarids, and other Nematocera.

Included as insects of interest are adults and nymphs of the ordersHemiptera and Homoptera such as, but not limited to, adelgids from thefamily Adelgidae, plant bugs from the family Miridae, cicadas from thefamily Cicadidae, leafhoppers, Empoasca spp.; from the familyCicadellidae, planthoppers from the families Cixiidae, Flatidae,Fulgoroidea, Issidae and Delphacidae, treehoppers from the familyMembracidae, psyllids from the family Psyllidae, whiteflies from thefamily Aleyrodidae, aphids from the family Aphididae, phylloxera fromthe family Phylloxeridae, mealybugs from the family Pseudococcidae,scales from the families Asterolecanidae, Coccidae, Dactylopiidae,Diaspididae, Eriococcidae Ortheziidae, Phoenicococcidae andMargarodidae, lace bugs from the family Tingidae, stink bugs from thefamily Pentatomidae, cinch bugs, Blissus spp.; and other seed bugs fromthe family Lygaeidae, spittlebugs from the family Cercopidae squash bugsfrom the family Coreidae and red bugs and cotton stainers from thefamily Pyrrhocoridae.

Agronomically important members from the order Homoptera furtherinclude, but are not limited to: Acyrthisiphon pisum Harris (pea aphid);Aphis craccivora Koch (cowpea aphid); A. fabae Scopoli (black beanaphid); A. gossypii Glover (cotton aphid, melon aphid); A. maidiradicisForbes (corn root aphid); A. pomi De Geer (apple aphid); A. spiraecolaPatch (spirea aphid); Aulacorthum solani Kaltenbach (foxglove aphid);Chaetosiphon fragaefolii Cockerell (strawberry aphid); Diuraphis noxiaKurdjumov/Mordvilko (Russian wheat aphid); Dysaphis plantagineaPaaserini (rosy apple aphid); Eriosoma lanigerum Hausmann (woolly appleaphid); Brevicoryne brassicae Linnaeus (cabbage aphid); Hyalopteruspruni Geoffroy (mealy plum aphid); Lipaphis erysimi Kaltenbach (turnipaphid); Metopolophium dirrhodum Walker (cereal aphid); Macrosiphumeuphorbiae Thomas (potato aphid); Myzus persicae Sulzer (peach-potatoaphid, green peach aphid); Nasonovia ribisnigri Mosley (lettuce aphid);Pemphigus spp. (root aphids and gall aphids); Rhopalosiphum maidis Fitch(corn leaf aphid); R. padi Linnaeus (bird cherry-oat aphid); Schizaphisgraminum Rondani (greenbug); Sipha flava Forbes (yellow sugarcaneaphid); Sitobion avenae Fabricius (English grain aphid); Therioaphismaculata Buckton (spotted alfalfa aphid); Toxoptera aurantii Boyer deFonscolombe (black citrus aphid) and T. citricida Kirkaldy (brown citrusaphid); Melanaphis sacchari (sugarcane aphid); Adelges spp. (adelgids);Phylloxera devastatrix Pergande (pecan phylloxera); Bemisia tabaciGennadius (tobacco whitefly, sweetpotato whitefly); B. argentifoliiBellows & Perring (silverleaf whitefly); Dialeurodes citri Ashmead(citrus whitefly); Trialeurodes abutiloneus (bandedwinged whitefly) andT. vaporariorum Westwood (greenhouse whitefly); Empoasca fabae Harris(potato leafhopper); Laodelphax striatellus Fallen (smaller brownplanthopper); Macrolestes quadrilineatus Forbes (aster leafhopper);Nephotettix cinticeps Uhler (green leafhopper); N. nigropictus Stal(rice leafhopper); Nilaparvata lugens Stal (brown planthopper);Peregrinus maidis Ashmead (corn planthopper); Sogatella furciferaHorvath (white-backed planthopper); Sogatodes orizicola Muir (ricedelphacid); Typhlocyba pomaria McAtee (white apple leafhopper);Erythroneoura spp. (grape leafhoppers); Magicicada septendecim Linnaeus(periodical cicada); Icerya purchasi Maskell (cottony cushion scale);Quadraspidiotus perniciosus Comstock (San Jose scale); Planococcus citriRisso (citrus mealybug); Pseudococcus spp. (other mealybug complex);Cacopsylla pyricola Foerster (pear psylla); Trioza diospyri Ashmead(persimmon psylla).

Agronomically important species of interest from the order Hemipterainclude, but are not limited to: Acrosternum hilare Say (green stinkbug); Anasa tristis De Geer (squash bug); Blissus leucopterusleucopterus Say (chinch bug); Corythuca gossypii Fabricius (cotton lacebug); Cyrtopeltis modesta Distant (tomato bug); Dysdercus suturellusHerrich-Schäffer (cotton stainer); Euschistus servus Say (brown stinkbug); E. variolarius Palisot de Beauvois (one-spotted stink bug);Graptostethus spp. (complex of seed bugs); Leptoglossus corculus Say(leaf-footed pine seed bug); Lygus lineolaris Palisot de Beauvois(tarnished plant bug); L. Hesperus Knight (Western tarnished plant bug);L. pratensis Linnaeus (common meadow bug); L. rugulipennis Poppius(European tarnished plant bug); Lygocoris pabulinus Linnaeus (commongreen capsid); Nezara viridula Linnaeus (southern green stink bug);Oebalus pugnax Fabricius (rice stink bug); Oncopeltus fasciatus Dallas(large milkweed bug); Pseudatomoscelis seriatus Reuter (cottonfleahopper).

Furthermore, embodiments may be effective against Hemiptera such,Calocoris norvegicus Gmelin (strawberry bug); Orthops campestrisLinnaeus; Plesiocoris rugicollis Fallen (apple capsid); Cyrtopeltismodestus Distant (tomato bug); Cyrtopeltis notatus Distant (suckfly);Spanagonicus albofasciatus Reuter (whitemarked fleahopper); Diaphnocorischlorionis Say (honeylocust plant bug); Labopidicola allii Knight (onionplant bug); Pseudatomoscelis seriatus Reuter (cotton fleahopper);Adelphocoris rapidus Say (rapid plant bug); Poecilocapsus lineatusFabricius (four-lined plant bug); Nysius ericae Schilling (false chinchbug); Nysius raphanus Howard (false chinch bug); Nezara viridulaLinnaeus (Southern green stink bug); Eurygaster spp.; Coreidae spp.;Pyrrhocoridae spp.; Tinidae spp.; Blostomatidae spp.; Reduviidae spp.and Cimicidae spp.

Also included are adults and larvae of the order Acari (mites) such asAceria tosichella Keifer (wheat curl mite); Petrobia latens Müller(brown wheat mite); spider mites and red mites in the familyTetranychidae, Panonychus ulmi Koch (European red mite); Tetranychusurticae Koch (two spotted spider mite); (T. mcdanieli McGregor (McDanielmite); T. cinnabarinus Boisduval (carmine spider mite); T. turkestaniUgarov & Nikolski (strawberry spider mite); flat mites in the familyTenuipalpidae, Brevipalpus lewisi McGregor (citrus flat mite); rust andbud mites in the family Eriophyidae and other foliar feeding mites andmites important in human and animal health, i.e., dust mites in thefamily Epidermoptidae, follicle mites in the family Demodicidae, grainmites in the family Glycyphagidae, ticks in the order Ixodidae. Ixodesscapularis Say (deer tick); I. holocyclus Neumann (Australian paralysistick); Dermacentor variabilis Say (American dog tick); Amblyommaamericanum Linnaeus (lone star tick) and scab and itch mites in thefamilies Psoroptidae, Pyemotidae and Sarcoptidae.

Insect pests of the order Thysanura are of interest, such as Lepismasaccharina Linnaeus (silverfish); Thermobia domestica Packard(firebrat).

Additional arthropod pests covered include: spiders in the order Araneaesuch as Loxosceles reclusa Gertsch and Mulaik (brown recluse spider) andthe Latrodectus mactans Fabricius (black widow spider) and centipedes inthe order Scutigeromorpha such as Scutigera coleoptrata Linnaeus (housecentipede).

Insect pest of interest include the superfamily of stink bugs and otherrelated insects including but not limited to species belonging to thefamily Pentatomidae (Nezara viridula, Halyomorpha halys, Piezodorusguildini, Euschistus servus, Acrosternum hilare, Euschistus heros,Euschistus tristigmus, Acrosternum hilare, Dichelops furcatus, Dichelopsmelacanthus, and Bagrada hilaris (Bagrada Bug)), the family Plataspidae(Megacopta cribraria—Bean plataspid) and the family Cydnidae(Scaptocoris castanea—Root stink bug) and Lepidoptera species includingbut not limited to: diamond-back moth, e.g., Helicoverpa zea Boddie;soybean looper, e.g., Pseudoplusia includens Walker and velvet beancaterpillar e.g., Anticarsia gemmatalis Hübner.

Methods for measuring pesticidal activity are well known in the art.See, for example, Czapla and Lang, (1990) J. Econ. Entomol.83:2480-2485; Andrews, et al., (1988) Biochem. J. 252:199-206; Marrone,et al., (1985) J. of Economic Entomology 78:290-293 and U.S. Pat. No.5,743,477, all of which are herein incorporated by reference in theirentirety. Generally, the protein is mixed and used in feeding assays.See, for example Marrone, et al., (1985) J. of Economic Entomology78:290-293. Such assays can include contacting plants with one or morepests and determining the plant's ability to survive and/or cause thedeath of the pests.

Nematodes include parasitic nematodes such as root-knot, cyst and lesionnematodes, including Heterodera spp., Meloidogyne spp. and Globoderaspp.; particularly members of the cyst nematodes, including, but notlimited to, Heterodera glycines (soybean cyst nematode); Heteroderaschachtii (beet cyst nematode); Heterodera avenae (cereal cyst nematode)and Globodera rostochiensis and Globodera pailida (potato cystnematodes). Lesion nematodes include Pratylenchus spp.

Seed Treatment

To protect and to enhance yield production and trait technologies, seedtreatment options can provide additional crop plan flexibility and costeffective control against insects, weeds and diseases. Seed material canbe treated, typically surface treated, with a composition comprisingcombinations of chemical or biological herbicides, herbicide safeners,insecticides, fungicides, germination inhibitors and enhancers,nutrients, plant growth regulators and activators, bactericides,nematocides, avicides and/or molluscicides. These compounds aretypically formulated together with further carriers, surfactants orapplication-promoting adjuvants customarily employed in the art offormulation. The coatings may be applied by impregnating propagationmaterial with a liquid formulation or by coating with a combined wet ordry formulation. Examples of the various types of compounds that may beused as seed treatments are provided in The Pesticide Manual: A WorldCompendium, C.D.S. Tomlin Ed., Published by the British Crop ProductionCouncil, which is hereby incorporated by reference.

Some seed treatments that may be used on crop seed include, but are notlimited to, one or more of abscisic acid, acibenzolar-S-methyl,avermectin, amitrol, azaconazole, azospirillum, azadirachtin,azoxystrobin, Bacillus spp. (including one or more of cereus, firmus,megaterium, pumilis, sphaericus, subtilis and/or thuringiensis species),bradyrhizobium spp. (including one or more of betae, canariense,elkanii, iriomotense, japonicum, liaonigense, pachyrhizi and/oryuanmingense), captan, carboxin, chitosan, clothianidin, copper,cyazypyr, difenoconazole, etidiazole, fipronil, fludioxonil,fluoxastrobin, fluquinconazole, flurazole, fluxofenim, harpin protein,imazalil, imidacloprid, ipconazole, isoflavenoids,lipo-chitooligosaccharide, mancozeb, manganese, maneb, mefenoxam,metalaxyl, metconazole, myclobutanil, PCNB, penflufen, penicillium,penthiopyrad, permethrine, picoxystrobin, prothioconazole,pyraclostrobin, rynaxypyr, S-metolachlor, saponin, sedaxane, TCMTB,tebuconazole, thiabendazole, thiamethoxam, thiocarb, thiram,tolclofos-methyl, triadimenol, trichoderma, trifloxystrobin,triticonazole and/or zinc. PCNB seed coat refers to EPA RegistrationNumber 00293500419, containing quintozen and terrazole. TCMTB refers to2-(thiocyanomethylthio) benzothiazole.

Seed varieties and seeds with specific transgenic traits may be testedto determine which seed treatment options and application rates maycomplement such varieties and transgenic traits in order to enhanceyield. For example, a variety with good yield potential but head smutsusceptibility may benefit from the use of a seed treatment thatprovides protection against head smut, a variety with good yieldpotential but cyst nematode susceptibility may benefit from the use of aseed treatment that provides protection against cyst nematode, and soon. Likewise, a variety encompassing a transgenic trait conferringinsect resistance may benefit from the second mode of action conferredby the seed treatment, a variety encompassing a transgenic traitconferring herbicide resistance may benefit from a seed treatment with asafener that enhances the plants resistance to that herbicide, etc.Further, the good root establishment and early emergence that resultsfrom the proper use of a seed treatment may result in more efficientnitrogen use, a better ability to withstand drought and an overallincrease in yield potential of a variety or varieties containing acertain trait when combined with a seed treatment.

Methods for Killing an Insect Pest and Controlling an Insect Population

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of a recombinant PIP-72 polypeptide. In some embodiments methodsare provided for killing an insect pest, comprising contacting theinsect pest with an insecticidally-effective amount of a recombinantpesticidal protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, anyone of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772 orSEQ ID NO: 852 or a variant thereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant PIP-72 polypeptide. Insome embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant pesticidal protein ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO:32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO:825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772 or SEQ ID NO: 852 ora variant thereof. As used herein, “controlling a pest population” or“controls a pest” refers to any effect on a pest that results inlimiting the damage that the pest causes. Controlling a pest includes,but is not limited to, killing the pest, inhibiting development of thepest, altering fertility or growth of the pest in such a manner that thepest provides less damage to the plant, decreasing the number ofoffspring produced, producing less fit pests, producing pests moresusceptible to predator attack or deterring the pests from eating theplant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population with an insecticidally-effective amount of arecombinant PIP-72 polypeptide. In some embodiments methods are providedfor controlling an insect pest population resistant to a pesticidalprotein, comprising contacting the insect pest population with aninsecticidally-effective amount of a recombinant pesticidal protein ofSEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10,SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO:32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO:825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772 or SEQ ID NO: 852 ora variant thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding a PIP-72 polypeptide. In someembodiments methods are provided for protecting a plant from an insectpest, comprising expressing in the plant or cell thereof a recombinantpolynucleotide encoding pesticidal protein of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO:528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO:771, SEQ ID NO: 772 or SEQ ID NO: 852 or variants thereof.

In some embodiments methods are provided for killing an insect pest,comprising contacting the insect pest with an insecticidally-effectiveamount of a recombinant polypeptide of SEQ ID NO: 20, SEQ ID NO: 22, SEQID NO: 24, SEQ I NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36,SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO: 937, SEQ IDNO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQ ID NO: 948 or a variantthereof.

In some embodiments methods are provided for controlling an insect pestpopulation, comprising contacting the insect pest population with aneffective amount of a recombinant polypeptide of SEQ ID NO: 20, SEQ IDNO: 22, SEQ ID NO: 24, SEQ I NO: 26, SEQ ID NO: 30, SEQ ID NO: 34, SEQID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931, SEQ ID NO:937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQ ID NO: 948or a variant thereof. As used herein, “controlling a pest population” or“controls a pest” refers to any effect on a pest that results inlimiting the damage that the pest causes. Controlling a pest includes,but is not limited to, killing the pest, inhibiting development of thepest, altering fertility or growth of the pest in such a manner that thepest provides less damage to the plant, decreasing the number ofoffspring produced, producing less fit pests, producing pests moresusceptible to predator attack or deterring the pests from eating theplant.

In some embodiments methods are provided for controlling an insect pestpopulation resistant to a pesticidal protein, comprising contacting theinsect pest population with an effective amount of a recombinantpolypeptide of SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ I NO:26, SEQ ID NO: 30, SEQ ID NO: 34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ IDNO: 930, SEQ ID NO: 931, SEQ ID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942,SEQ ID NO: 947, or SEQ ID NO: 948 or a variant thereof.

In some embodiments methods are provided for protecting a plant from aninsect pest, comprising expressing in the plant or cell thereof arecombinant polynucleotide encoding a pesticidal protein of SEQ ID NO:20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ I NO: 26, SEQ ID NO: 30, SEQ IDNO: 34, SEQ ID NO: 36, SEQ ID NO: 929, SEQ ID NO: 930, SEQ ID NO: 931,SEQ ID NO: 937, SEQ ID NO: 938, SEQ ID NO: 942, SEQ ID NO: 947, or SEQID NO: 948 or variants thereof.

Insect Resistance Management (IRM) Strategies

Expression of B. thuringiensis δ-endotoxins in transgenic corn plantshas proven to be an effective means of controlling agriculturallyimportant insect pests (Perlak, et al., 1990; 1993). However, insectshave evolved that are resistant to B. thuringiensis δ-endotoxinsexpressed in transgenic plants. Such resistance, should it becomewidespread, would clearly limit the commercial value of germplasmcontaining genes encoding such B. thuringiensis δ-endotoxins.

One way to increasing the effectiveness of the transgenic insecticidesagainst target pests and contemporaneously reducing the development ofinsecticide-resistant pests is to use provide non-transgenic (i.e.,non-insecticidal protein) refuges (a section of non-insecticidalcrops/corn) for use with transgenic crops producing a singleinsecticidal protein active against target pests. The United StatesEnvironmental Protection Agency(epa.gov/oppbppdl/biopesticides/pips/bt_corn_refuge_2006.htm, which canbe accessed using the www prefix) publishes the requirements for usewith transgenic crops producing a single Bt protein active againsttarget pests. In addition, the National Corn Growers Association, ontheir website:(ncga.com/insect-resistance-management-fact-sheet-bt-corn, which can beaccessed using the www prefix) also provides similar guidance regardingrefuge requirements. Due to losses to insects within the refuge area,larger refuges may reduce overall yield.

Another way of increasing the effectiveness of the transgenicinsecticides against target pests and contemporaneously reducing thedevelopment of insecticide-resistant pests would be to have a repositoryof insecticidal genes that are effective against groups of insect pestsand which manifest their effects through different modes of action.

Expression in a plant of two or more insecticidal compositions toxic tothe same insect species, each insecticide being expressed at efficaciouslevels would be another way to achieve control of the development ofresistance. This is based on the principle that evolution of resistanceagainst two separate modes of action is far more unlikely than only one.Roush, for example, outlines two-toxin strategies, also called“pyramiding” or “stacking,” for management of insecticidal transgeniccrops. (The Royal Society. Phil. Trans. R. Soc. Lond. B. (1998)353:1777-1786). Stacking or pyramiding of two different proteins eacheffective against the target pests and with little or nocross-resistance can allow for use of a smaller refuge. The USEnvironmental Protection Agency requires significantly less (generally5%) structured refuge of non-Bt corn be planted than for single traitproducts (generally 20%). There are various ways of providing the IRMeffects of a refuge, including various geometric planting patterns inthe fields and in-bag seed mixtures, as discussed further by Roush.

In some embodiments the PIP-72 polypeptides of the disclosure are usefulas an insect resistance management strategy in combination (i.e.,pyramided) with other pesticidal proteins include but are not limited toBt toxins, Xenorhabdus sp. or Photorhabdus sp. insecticidal proteins,and the like.

Provided are methods of controlling Lepidoptera and/or Coleoptera insectinfestation(s) in a transgenic plant that promote insect resistancemanagement, comprising expressing in the plant at least two differentinsecticidal proteins having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprise a PIP-72 polypeptide insecticidal to insects in the orderLepidoptera and/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management the at least one of the insecticidal proteinscomprises a protein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ IDNO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, anyone of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772,SEQ ID NO: 852, any one of SEQ ID NO: 903-SEQ ID NO: 914, SEQ ID NO:927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO:941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946or variants thereof, insecticidal to insects in the order Lepidopteraand/or Coleoptera.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise expressing in the transgenic plant aPIP-72 polypeptide and a Cry protein insecticidal to insects in theorder Lepidoptera and/or Coleoptera having different modes of action.

In some embodiments the methods of controlling Lepidoptera and/orColeoptera insect infestation in a transgenic plant and promoting insectresistance management comprise in the transgenic plant a protein of SEQID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32,any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO: 852, any one ofSEQ ID NO: 903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ IDNO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936,SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ IDNO: 944, SEQ ID NO: 945, or SEQ ID NO: 946 or variants thereof and a Cryprotein insecticidal to insects in the order Lepidoptera and/orColeoptera having different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of a PIP-72 polypeptide insecticidal tothe insect species in combination with a second insecticidal protein tothe insect species having different modes of action.

Also provided are methods of reducing likelihood of emergence ofLepidoptera and/or Coleoptera insect resistance to transgenic plantsexpressing in the plants insecticidal proteins to control the insectspecies, comprising expression of a protein of SEQ ID NO: 2, SEQ ID NO:4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO:14, SEQ ID NO: 18, SEQ ID NO: 28, SEQ ID NO: 32, any one of SEQ ID NO:528-SEQ ID NO: 768, any one of SEQ ID NO: 825-SEQ ID NO: 844, SEQ ID NO:771, SEQ ID NO: 772, SEQ ID NO: 852, any one of SEQ ID NO: 903-SEQ IDNO: 914, SEQ ID NO: 927, SEQ ID NO: 928, SEQ ID NO: 932, SEQ ID NO: 933,SEQ ID NO: 934, SEQ ID NO: 935, SEQ ID NO: 936, SEQ ID NO: 939, SEQ IDNO: 940, SEQ ID NO: 941, SEQ ID NO: 943, SEQ ID NO: 944, SEQ ID NO: 945,or SEQ ID NO: 946 or variants thereof, insecticidal to the insectspecies in combination with a second insecticidal protein to the insectspecies having different modes of action.

Also provided are means for effective Lepidoptera and/or Coleopterainsect resistance management of transgenic plants, comprisingco-expressing at high levels in the plants two or more insecticidalproteins toxic to Lepidoptera and/or Coleoptera insects but eachexhibiting a different mode of effectuating its killing activity,wherein the two or more insecticidal proteins comprise a PIP-72polypeptide and a Cry protein. Also provided are means for effectiveLepidoptera and/or Coleoptera insect resistance management of transgenicplants, comprising co-expressing at high levels in the plants two ormore insecticidal proteins toxic to Lepidoptera and/or Coleopterainsects but each exhibiting a different mode of effectuating its killingactivity, wherein the two or more insecticidal proteins comprise aprotein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28,SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772, SEQ ID NO:852, any one of SEQ ID NO: 903-SEQ ID NO: 914, SEQ ID NO: 927, SEQ IDNO: 928, SEQ ID NO: 932, SEQ ID NO: 933, SEQ ID NO: 934, SEQ ID NO: 935,SEQ ID NO: 936, SEQ ID NO: 939, SEQ ID NO: 940, SEQ ID NO: 941, SEQ IDNO: 943, SEQ ID NO: 944, SEQ ID NO: 945, or SEQ ID NO: 946 or variantsthereof and a Cry protein.

In addition, methods are provided for obtaining regulatory approval forplanting or commercialization of plants expressing proteins insecticidalto insects in the order Lepidoptera and/or Coleoptera, comprising thestep of referring to, submitting or relying on insect assay binding datashowing that the PIP-72 polypeptide does not compete with binding sitesfor Cry proteins in such insects. In addition, methods are provided forobtaining regulatory approval for planting or commercialization ofplants expressing proteins insecticidal to insects in the orderLepidoptera and/or Coleoptera, comprising the step of referring to,submitting or relying on insect assay binding data showing that theprotein of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 18, SEQ ID NO: 28,SEQ ID NO: 32, any one of SEQ ID NO: 528-SEQ ID NO: 768, any one of SEQID NO: 825-SEQ ID NO: 844, SEQ ID NO: 771, SEQ ID NO: 772 or SEQ ID NO:852 or variant thereof does not compete with binding sites for Cryproteins in such insects.

Methods for Increasing Plant Yield

Methods for increasing plant yield are provided. The methods compriseproviding a plant or plant cell expressing a polynucleotide encoding thepesticidal polypeptide sequence disclosed herein and growing the plantor a seed thereof in a field infested with a pest against which thepolypeptide has pesticidal activity. In some embodiments, thepolypeptide has pesticidal activity against a Lepidopteran, Coleopteran,Dipteran, Hemipteran or nematode pest, and the field is infested with aLepidopteran, Hemipteran, Coleopteran, Dipteran or nematode pest.

As defined herein, the “yield” of the plant refers to the quality and/orquantity of biomass produced by the plant. “Biomass” as used hereinrefers to any measured plant product. An increase in biomass productionis any improvement in the yield of the measured plant product.Increasing plant yield has several commercial applications. For example,increasing plant leaf biomass may increase the yield of leafy vegetablesfor human or animal consumption. Additionally, increasing leaf biomasscan be used to increase production of plant-derived pharmaceutical orindustrial products. An increase in yield can comprise any statisticallysignificant increase including, but not limited to, at least a 1%increase, at least a 3% increase, at least a 5% increase, at least a 10%increase, at least a 20% increase, at least a 30%, at least a 50%, atleast a 70%, at least a 100% or a greater increase in yield compared toa plant not expressing the pesticidal sequence.

In specific methods, plant yield is increased as a result of improvedpest resistance of a plant expressing a PIP-72 polypeptide disclosedherein. Expression of the PIP-72 polypeptide results in a reducedability of a pest to infest or feed on the plant, thus improving plantyield.

Methods of Processing

Further provided are methods of processing a plant, plant part or seedto obtain a food or feed product from a plant, plant part or seedcomprising a PIP-72 polypeptide. The plants, plant parts or seedsprovided herein, can be processed to yield oil, protein products and/orby-products that are derivatives obtained by processing that havecommercial value. Non-limiting examples include transgenic seedscomprising a nucleic acid molecule encoding a PIP-72 polypeptide whichcan be processed to yield soy oil, soy products and/or soy by-products.

“Processing” refers to any physical and chemical methods used to obtainany soy product and includes, but is not limited to, heat conditioning,flaking and grinding, extrusion, solvent extraction or aqueous soakingand extraction of whole or partial seeds

The following examples are offered by way of illustration and not by wayof limitation.

EXPERIMENTALS Example 1—Identification of a Insecticidal Protein ActiveAgainst Western Corn Root Worm (WCRW) from Strain SS143D5

The WCRW (Diabrotica virgifera virgifera) active protein PIP-72Aa wasidentified by protein purification, liquid chromatography massspectrometry (LC-MS/MS) and PCR cloning from Pseudomonas chlororaphisstrain SS143D5 as follows:

Insecticidal activity against WCRW (Diabrotica virgifera virgifera) wasobserved from a clear cell lysate of SS143D5 grown in Trypticase soymedium (Tryptone 17 g/L, enzymatic digest of soy meal 3 g/L, Dextrose2.5 g/L, Sodium Chloride 5 g/L, K2HPO4 2.5 g/L) and cultured overnightat 2600 with shaking at 250 rpm. This insecticidal activity exhibitedheat and proteinase sensitivity indicating proteinaceous nature. Forprotein extraction, cells were thawed and re-suspended in 50 mM sodiumacetate buffer, pH 5 (buffer A) containing protease inhibitor cocktail Vfrom CalBiochem. A crude cleared lysate was obtained by passing thecells through a homogenizer at 30,000 psi, followed by centrifugation at13,800×g for 20 min.

WCRW bioassays were conducted using 10 microliter cell lysate samplesmixed with molten low-melt WCRW diet (Southland Products Inc., LakeVillage, Ark.) in a 96 well format. Diabrotica virgifera virgiferaneonates were placed into each well of a 96 well plate. The assay wasrun for 4 days at 25° C. and then was scored for insect mortality andstunting of insect growth. The scores were noted as dead, severelystunted (little or no growth but alive), stunted (growth to secondinstar but not equivalent to controls) or no activity.

Genomic DNA from strain SS143D5 was extracted with a Sigma-Aldrich®Bacterial Genomic DNA Extraction Kit (Cat # NA2110-KT, Sigma-Aldrich, POBox 14508, St. Louis, Mo. 63178) according to the manufactures'instructions. The DNA concentration was determined using a NanoDrop®Spectrophotometer (Thermo Fisher Scientific®, 3411 Silverside Road,Bancroft Building, Suite 100, Willmington, Del. 19810) and the genomicDNA was diluted to 40 ng/ul with sterile water. A 25 ul PCR reaction wasset up by combining 80 ng genomic DNA, 2 ul (5 uM) 16S ribosomal DNAprimers TACCTTGTTACGACTT (SEQ ID NO: 285) and AGAGTTTGATCMTGGCTCAG (SEQID NO: 286), 1 ul 10 cmM dNTP, 1× Phusion® HF™ buffer, and 1 unit ofPhusion® High-Fidelity DNA Polymerase (New England Biolabs®, Cat#M0530L, 240 County Road, Ipswich, Mass. 01938-2723). The PCR reactionwas run in a MJ Research PTC-200 Thermo Cycler (Bio-Rad® Laboratories,Inc., 1000 Alfred Nobel Drive, Hercules, Calif., 94547, USA) with thefollowing program: 96° C. 1 min; 30 cycles of 96° C. 15 seconds, 52° C.2 minutes and 72° C. 2 minutes; 72° C. 10 minutes; and hold on 4° C. ThePCR products were purified with QiaQuick® DNA purification Kit (Cat#28104, QIAGEN® Inc., 27220 Turnberry Lane, Valencia, Calif. 91355). Thepurified PCR sample was DNA sequenced and the resulting 16S ribosomalDNA sequence was BLAST searched against the NCBI database whichindicated that SS143D5 is a Pseudomonas chlororaphis strain. ThePseudomonas chlororaphis strain SS143D5 was deposited on Feb. 7, 2013under accession # NRRL B-50810 with the Agricultural Research ServiceCulture Collection (NRRL), 1815 North University Street, Peoria, Ill.61604, (nrrl.ncaur.usda.gov, which can be accessed on the world-wide webusing the “www” prefix).

Isolated strain SS143D5 genomic DNA was also prepared according to alibrary construction protocol developed by Illumina and sequenced usingthe Illumina® Genome Analyzer® IIx (Cat# SY-301-1301, Illumina Inc.,9885 Towne Center Drive, San Diego, Calif. 92121). The nucleic acidcontig sequences were assembled and open reading frames were generated.

Cell pellet of an overnight culture of SS143D5 grown in 2×YT broth at26° C. with shaking at 250 rpm was lyzed at ˜20,000 psi afterresuspension in acetate buffer, pH 5. The crude lysate was cleared bycentrifugation and loaded onto a HiTrap™ S-HP column (GE Healthcare, 800Centennial Avenue, P.O. Box 1327, Piscataway, N.J. 08855). Bound proteinwas eluted with a linear sodium chloride gradient and fractionated.Fractions containing protein of interest were pooled and bufferexchanged for loading onto a MonoQ™ column (GE Healthcare), run at pH 8.PIP-72Aa (SEQ ID NO: 2) was eluted with a linear sodium chloridegradient and after activity confirmation further purified by hydrophobicinteraction chromatography. For this the protein was adjusted to 0.8 Mammonium sulfate, loaded onto a HiTrap™ Butyl-HP column (GE Healthcare)and active protein was recovered in the unbound fraction. SDS-PAGEanalysis showed a single band after staining with Coomassie™ Blue dye.The protein band was excised, digested with trypsin and analyzed bynano-liquid chromatography/electrospray tandem mass spectrometry(nano-LC/ESI-MS/MS) on a Thermo Q Exactive™ Orbitrap™ mass spectrometer(Thermo Fisher Scientific®, 81 Wyman Street, Waltham, Mass. 02454)interfaced with an Eksigent NanoLC 1-D Plus nano-lc system (AB Sciex™,500 Old Connecticut Path, Framingham, Mass. 01701, USA). Ten product ionspectra were collected in an information dependent acquisition modeafter a MS1 survey scan.

Protein identification was done by database searches using Mascot®(Matrix Science, 10 Perrins Lane, London NW3 1QY, UK). The searchagainst the in-house database Bacteria-Plus, which combines allbacterial protein sequences and keratin sequences derived from the NCBInon-redundant database (nr) as well as in-house protein sequences,identified a novel gene encoded by strain SS143D5, which was designatedas PIP-72Aa (SEQ ID NO: 1).

Example 2—Identification of Homologs of PIP-72Aa

Gene identities may be determined by conducting BLAST (Basic LocalAlignment Search Tool; Altschul, et al., (1993) J. Mol. Biol.215:403-410; see also ncbi.nlm.nih.gov/BLAST/, which can be accessedusing the www prefix) searches under default parameters for similarityto sequences contained in the publically available BLAST “nr” database(comprising all non-redundant GenBank CDS translations, sequencesderived from the 3-dimensional structure Brookhaven Protein Data Bank,the last major release of the 25 SWISS-PROT protein sequence database,EMBL, and DDBJ databases. In addition to public databases, internalDuPont Pioneer databases were searched. The polynucleotide sequences SEQID NO: 1 was analyzed.

The search identified several homologs of PIP-72Aa (SEQ ID NO: 2) havingvarying percent identity to PIP-72Aa (SEQ ID NO: 2). Insecticidal activePIP-72Aa homologs designated herein as: PIP-72Ba (SEQ ID NO: 4);PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ ID NO: 8); PIP-72 Da (SEQ ID NO:10); PIP-72Db (SEQ ID NO: 12); PIP-72Dc (SEQ ID NO: 14); PIP-72Fa (SEQID NO: 18); PIP-72Ff (SEQ ID NO: 28) and PIP-72Gb (SEQ ID NO: 32) wereidentified from a DuPont Pioneer internal bacterial genome databasefrom: Pseudomonas rhodesiae; Pseudomonas chlororaphis; Pseudomonasmandelii; Pseudomonas congelans; Pseudomonas mandelii; Pseudomonasficuserectae; Pseudomonas mosselii; Pseudomonas chlororaphis; andPseudomonas chlororaphis respectively. PIP-72Ba (SEQ ID NO: 4); PIP-72Ca(SEQ ID NO: 6); PIP-72Cb (SEQ ID NO: 8); PIP-72 Da (SEQ ID NO: 10);PIP-72Db (SEQ ID NO: 12); PIP-72Dc (SEQ ID NO: 14); PIP-72Fa (SEQ ID NO:18); PIP-72Ff (SEQ ID NO: 28) and PIP-72Gb (SEQ ID NO: 32) are encodedby SEQ ID NO: 3; SEQ ID NO: 5; SEQ ID NO: 7; SEQ ID NO: 9; SEQ ID NO:11; SEQ ID NO: 13; SEQ ID NO: 17; SEQ ID NO: 27 and SEQ ID NO: 31,respectively. The inactive low identity homologs designated herein asPIP-72Ea (SEQ ID NO: 16) and PIP-72Ge (SEQ ID NO: 38) were identifiedfrom a DuPont Pioneer internal bacterial genome database fromPseudomonas congelans and Pseudomonas chlororaphis respectively.PIP-72Ea and PIP-72Ge are encoded by SEQ ID NO: 15 and SEQ ID NO: 37,respectively.

The more distant insecticidal active homologs designated herein as:GBP_A3175 (SEQ ID NO: 20—Hypothetical Protein GBP346_A3175, Accession #YP_002897852); JG43047 (SEQ ID NO: 24—Hypothetical Protein, Accession #JGI-2165143047) and PFL_6283 (SEQ ID NO: 30—Hypothetical Protein,Accession # YP_004842361.1) were identified from external publicdatabases from: Burkholderia pseudomallei MSHR346; Pseudomonas sp. andPseudomonas protegens Pf-5, respectively. GBP_A3175 (SEQ ID NO: 20);JG43047 (SEQ ID NO: 24) and PFL_6283 (SEQ ID NO: 30) are encoded by SEQID NO: 19, SEQ ID NO: 23 and SEQ ID NO: 29, respectively.

The more distant homologs, which were not expressed or were inactive atthe concentrations tested were designated as SRBS_294080 (SEQ ID NO:22—Hypothetical Protein from a Switchgrass rhizospere MC, Accession #JGI 2021745495); SwiRh_4910 (SEQ ID NO: 26—Hypothetical Protein fromSwitchgrass rhizosphere, Accession # JGI-SwiRh_1014910); XBJ1_1078 (SEQID NO: 34—Hypothetical Protein XBJ1_1078 from Xenorhabdus bovieniiSS-2004, Accession # YP_003467009) and plu2373 (SEQ ID NO:36—Hypothetical Protein plu2373 from Photorhabdus luminescens subsp.laumondii TTO1, Accession # NP_929618) were identified from externalpublic databases; Xenorhabdus encoded by SEQ ID NO: 21; SEQ ID NO: 25;SEQ ID NO: 33 and SEQ ID NO: 35, respectively.

Additional PIP-72 homologs were identified from BLAST searching publicdatabases and internal DuPont Pioneer databases basically as describedabove. Table 5 shows the PIP-72 polypeptide designation, percentidientiy to IPDO72Aa (SEQ ID NO: 2), source of the bacterial strain, andbacterial species. Table 6 shows the PIP-72 polypeptide designation,polypeptide sequence identifier, and polynucleotide sequence identifier.

TABLE 5 Identity to PIP-72Aa PIP-72 SEQ ID homolog NO: 2 Source SpeciesPIP-72Ab 94.2% internal active strain— Pseudomonas SSP587D6-1chlororaphis PIP-72Bb 87.4% internal collection— Pseudomonas SSP555A5b;JH54973-1 brassicacearum WP_030131237 77.9% NCBI-WP_030131237—Pseudomonas hypothetical protein sp. QTF5 WP_016417435 50.6%NCBI-hypothetical Halomonas protein WP_016417435.1 anticariensisBDL_2850 41.1% NCBI hypothetical Burkholderia protein BDL_2850pseudomallei (AHE26696) PIP-72Fh 41.9% internal active strainPseudomonas SSP533D8d; SS143C5; entomophila SSP436A3a; SSP535F2a; L48SSP429C9a PIP-72Fi 41.9% internal active strain Pseudomonas SSP533G1aentomophila PIP-72Fj 39.5% internal active strain Pseudomonas JH44835-2;JH59153-1 chlororaphis PIP-72Fk 41.9% internal active strain PseudomonasJH52442-1; JH52448-2 chlororaphis PIP-72Fl 44.9% internal active strainBurkholderia JH59178-1; JH59094-1 multivorans BBK_2354 43.0% NCBIhypothetical Burkholderia protein WP_023360071.1 pseudomallei BBK_2354D512_15386 38.6% NCBI hypothetical Burkholderia protein D512_15386pseudomallei ZP_23811189.1 MSHR1043 PIP-72Gg   33% internal collection—Pseudomonas SSP459B9-3; SSP541A11-3; chlororaphis SSP560B2a; SSP560B12cPIP-72Gh 38.4% internal collection— Pseudomonas SSP469F1d chlororaphisPIP-72Gi 39.5% internal collection— Pseudomonas SSP471D1a; SSP471D1c;mosselli SSP4G8; SSP514E4-3; SSP514E2-1; BTI_1023 37.5% hypotheticalprotein Burkholderia BTI_1023, YP_007917531; thallandensisEMBL-N0ALH1_BURTH PIP-72Gk 37.5% internal collection— PseudomonasJH34931-1 protegens PIP-72Gl 39.5% internal collection— PseudomonasSSP490B11a; SSP514E2-1; plecoglossicida SSP341E4-3 PIP-72Gm 37.8%internal collection— JH23445-2 PIP-72Gn 38.4% internal collection—Pseudomonas SSP579D5-2, SSP581D7-1; chlororaphis SSP579D8-1 WP_02853664639.5% NCBI-WP_028536646.1— Paludibacterium hypothetical proteinyongneupense BTRA_1468 38.9% NCBI-AIC88674.1— Burkholderia hypotheticalprotein thallandensis BTRA_1468

TABLE 6 PIP-72 homolog Polypeptide Polynucleotide PIP-72Ab SEQ ID NO:927 SEQ ID NO: 949 PIP-72Bb SEQ ID NO: 928 SEQ ID NO: 950 WP_030131237SEQ ID NO: 929 SEQ ID NO: 951 WP_016417435 SEQ ID NO: 930 SEQ ID NO: 952BDL_2850 SEQ ID NO: 931 SEQ ID NO: 953 PIP-72Fh SEQ ID NO: 932 SEQ IDNO: 954 PIP-72Fi SEQ ID NO: 933 SEQ ID NO: 955 PIP-72Fj SEQ ID NO: 934SEQ ID NO: 956 PIP-72Fk SEQ ID NO: 935 SEQ ID NO: 957 PIP-72Fl SEQ IDNO: 936 SEQ ID NO: 958 BBK_2354 SEQ ID NO: 937 SEQ ID NO: 959 D512_15386SEQ ID NO: 938 SEQ ID NO: 960 PIP-72Gg SEQ ID NO: 939 SEQ ID NO: 961PIP-72Gh SEQ ID NO: 940 SEQ ID NO: 962 PIP-72Gi SEQ ID NO: 941 SEQ IDNO: 963 BTI_1023 SEQ ID NO: 942 SEQ ID NO: 964 PIP-72Gk SEQ ID NO: 943SEQ ID NO: 965 PIP-72Gl SEQ ID NO: 944 SEQ ID NO: 966 PIP-72Gm SEQ IDNO: 945 SEQ ID NO: 967 PIP-72Gn SEQ ID NO: 946 SEQ ID NO: 968WP_028536646 SEQ ID NO: 947 SEQ ID NO: 969 BTRA_1468 SEQ ID NO: 948 SEQID NO: 970

FIG. 1 shows the amino acid sequence alignment of PIP-72Aa (SEQ ID NO:2), PIP-72Ba (SEQ ID NO: 4); PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ IDNO: 8); PIP-72 Da (SEQ ID NO: 10); PIP-72Db (SEQ ID NO: 12); PIP-72Dc(SEQ ID NO: 14); PIP-72Ea (SEQ ID NO: 16), PIP-72Fa (SEQ ID NO: 18);GBP_A3175 (SEQ ID NO: 20), SRBS_294080 (SEQ ID NO: 22); JG43047 (SEQ IDNO: 24); SwiRh_4910 (SEQ ID NO: 26); PIP-72Ff (SEQ ID NO: 28), PFL_6283(SEQ ID NO: 30); PIP-72Gb (SEQ ID NO: 32); XBJ1_1078 (SEQ ID NO: 34);plu2373 (SEQ ID NO: 36); and PIP-72Ge (SEQ ID NO: 38). The amino acidsequence diversity is highlighted. Amino acids 37-51 (Motif 1) relativeto PIP-72Aa (SEQ ID NO: 2) are underlined.

FIG. 2 shows an alignment of the amino acid sequences of PIP-72Aa (SEQID NO: 2), PIP-72Ab (SEQ ID NO: 927); PIP-72Ba (SEQ ID NO: 4); PIP-72Bb(SEQ ID NO: 928); PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ ID NO: 8);WP_030131237 (SEQ ID NO: 929); PIP-72 Da (SEQ ID NO: 10); PIP-72Db (SEQID NO: 12); PIP-72Dc (SEQ ID NO: 14); PIP-72Fa (SEQ ID NO: 18); andGBP_A3175 (SEQ ID NO: 20). The amino acid sequence diversity betweenPIP-72Aa (SEQ ID NO: 2) and the other homologs is indicated withshading.

FIG. 3 shows the amino acid sequence alignment of PIP-72Aa (SEQ ID NO:2), PIP-72Ba (SEQ ID NO: 4); PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ IDNO: 8); PIP-72 Da (SEQ ID NO: 10); PIP-72Db (SEQ ID NO: 12); andPIP-72Dc (SEQ ID NO: 14). The amino acid sequence diversity betweenPIP-72Aa (SEQ ID NO: 2) and the other homologs is indicated withshading.

FIG. 4 shows the amino acid sequence alignment of WP_030131237 (SEQ IDNO: 929) PIP-72Ca (SEQ ID NO: 6); PIP-72Cb (SEQ ID NO: 8); PIP-72 Da(SEQ ID NO: 10); PIP-72Db (SEQ ID NO: 12); and PIP-72Dc (SEQ ID NO: 14).The amino acid sequence diversity between PIP-72Da (SEQ ID NO: 10) andthe other homologs is indicated with shading.

FIG. 5 shows an alignment of the amino acid sequences of PIP-72Fh (SEQID NO: 932), PIP-72Gi (SEQ ID NO: 941); PIP-72Fi (SEQ ID NO: 933);PIP-72GI (SEQ ID NO: 944); PIP-72Fa (SEQ ID NO: 14). The amino aciddiversity between PIP-72Ca (SEQ ID NO: 2) and the other homologs isindicated with shading.

Table 7 shows the sequence identity between the PIP-72 homologs.

TABLE 7 GBP_A3175 PIP-72Ab PIP-72Bb WP_030131237 WP_016417435 BDL_2850PIP-72Fh PIP-72Fi SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQID NO: 20 NO: 927 NO: 928 NO: 929 NO: 930 NO: 931 NO: 932 NO: 933PIP-72Aa 42.2 94.2 88.4 77.9 51.2 41.1 39.6 39.6 (SEQ ID NO: 2)GBP_A3175 — 43.3 46.7 46.7 47.8 97.8 34.4 38.7 PIP-72Ab — — 87.2 75.648.8 42.2 39.6 39.6 PIP-72Bb — — — 80.2 48.8 45.6 40.7 40.7 WP_030131237— — — — 52.3 45.6 37.4 37.4 WP_016417435 — — — — — 46.7 33.0 33.0BDL_2850 — — — — — — 33.3 37.6 PIP-72Fh — — — — — — — 93.4 PIP-72Fi — —— — — — — — PIP-72Fj — — — — — — — — PIP-72Fk — — — — — — — — PIP-72Fl —— — — — — — — BBK_2354 — — — — — — — — D512_15386 — — — — — — — —PIP-72Gg — — — — — — — — PIP-72Gh — — — — — — — — PIP-72Gi — — — — — — —— BTI_1023 — — — — — — — — PIP-72Gk — — — — — — — — PIP-72Gl — — — — — —— — PIP-72Gm — — — — — — — — PIP-72Gn — — — — — — — — WP_028536646 — — —— — — — — BTRA_1468 — — — — — — — — JG43047 — — — — — — — — PFL_6283 — —— — — — — — PIP-72Ba — — — — — — — — PIP-72Ca — — — — — — — — PIP-72Cb —— — — — — — — PIP-72Da — — — — — — — — PIP-72Db — — — — — — — — PIP-72Dc— — — — — — — — PIP-72Ea — — — — — — — — PIP-72Fa — — — — — — — —PIP-72Ff — — — — — — — — PIP-72Gb — — — — — — — — PIP-72Ge — — — — — — —— Plu2373 — — — — — — — — SRBS_294080 — — — — — — — — SwiRh_4910 — — — —— — — — XBJ1_1078 — — — — — — — — PIP-72Fj PIP-72Fk PIP-72Fl BBK_2354D512_15386 PIP-72Gg PIP-72Gh PIP-72Gi SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID SEQ ID NO: 934 NO: 935 NO: 936 NO: 937 NO: 938 NO: 939 NO:940 NO: 941 PIP-72Aa 35.4 37.5 44.9 41.1 37.0 33.7 34.4 37.4 (SEQ ID NO:2) GBP_A3175 38.1 41.2 42.9 98.9 89.1 34.7 38.1 37.6 PIP-72Ab 33.3 35.446.1 42.2 38.0 31.6 32.3 37.4 PIP-72Bb 31.2 33.3 43.8 45.6 41.3 33.730.2 38.5 WP_030131237 34.4 35.4 47.2 45.6 42.4 31.6 33.3 35.2WP_016417435 35.4 37.4 36.0 46.7 42.4 35.8 34.3 33.0 BDL_2850 38.1 41.241.8 98.9 91.3 33.7 38.1 36.6 PIP-72Fh 52.1 55.2 28.3 33.3 32.6 43.452.1 94.5 PIP-72Fi 53.1 56.2 27.2 37.6 36.8 41.7 53.1 94.5 PIP-72Fj —80.2 34.0 38.1 38.4 48.5 92.7 55.2 PIP-72Fk — — 32.0 41.2 39.4 42.6 80.258.3 PIP-72Fl — — — 41.8 37.6 35.4 36.1 29.3 BBK_2354 — — — — 90.2 34.738.1 36.6 D512_15386 — — — — — 33.7 38.4 35.8 PIP-72Gg — — — — — — 50.543.8 PIP-72Gh — — — — — — — 55.2 PIP-72Gi — — — — — — — — BTI_1023 — — —— — — — — PIP-72Gk — — — — — — — — PIP-72Gl — — — — — — — — PIP-72Gm — —— — — — — — PIP-72Gn — — — — — — — — WP_028536646 — — — — — — — —BTRA_1468 — — — — — — — — JG43047 — — — — — — — — PFL_6283 — — — — — — —— PIP-72Ba — — — — — — — — PIP-72Ca — — — — — — — — PIP-72Cb — — — — — —— — PIP-72Da — — — — — — — — PIP-72Db — — — — — — — — PIP-72Dc — — — — —— — — PIP-72Ea — — — — — — — — PIP-72Fa — — — — — — — — PIP-72Ff — — — —— — — — PIP-72Gb — — — — — — — — PIP-72Ge — — — — — — — — Plu2373 — — —— — — — — SRBS_294080 — — — — — — — — SwiRh_4910 — — — — — — — —XBJ1_1078 — — — — — — — — BTI_1023 PIP-72Gk PIP-72Gl PIP_72Gm PIP-72GnWP_028536646 BTRA_1468 JG43047 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID SEQ ID NO: 942 NO: 943 NO: 944 NO: 945 NO: 946 NO: 947 NO: 948NO: 24 PIP-72Aa 30.0 37.4 39.6 37.8 33.7 40.0 31.1 35.7 (SEQ ID NO: 2)GBP_A3175 77.8 43.2 37.6 39.1 37.4 34.4 77.8 37.4 PIP-72Ab 30.0 38.539.6 35.6 31.6 35.2 32.2 33.7 PIP-72Bb 32.2 39.6 40.7 36.7 29.6 36.733.3 31.6 WP_030131237 34.4 37.4 37.4 35.6 32.7 36.4 35.6 34.7WP_016417435 35.6 34.1 33.0 35.5 33.7 35.6 36.7 34.7 BDL_2850 80.0 42.136.6 38.0 37.4 34.4 80.0 37.4 PIP-72Fh 29.0 53.3 94.5 56.0 52.0 24.230.1 52.0 PIP-72Fi 32.3 53.3 98.9 56.0 54.1 24.2 33.3 53.1 PIP-72Fj 29.948.5 52.1 59.4 90.8 25.0 30.9 96.9 PIP-72Fk 33.0 50.5 55.2 53.1 77.621.9 35.4 79.6 PIP-72Fl 31.9 28.3 27.2 35.9 34.3 30.9 34.1 32.3 BBK_2354 78.9 43.2 36.6 39.1 37.4 33.3 78.9 37.4 D512_15386 73.9 41.2 35.836.2 37.6 29.3 73.9 37.6 PIP-72Gg 28.1 46.9 41.7 67.4 48.5 21.2 28.448.5 PIP-72Gh 29.9 48.5 52.1 59.4 91.8 24.0 30.9 89.8 PIP-72Gi 31.2 53.395.6 58.2 55.1 23.1 32.3 55.1 BTI_1023 — 34.7 31.2 30.4 29.3 28.1 94.729.3 PIP-72Gk — — 52.2 51.6 48.5 30.2 34.0 50.0 PIP-72Gl — — — 56.0 53.124.2 32.3 52.0 PIP-72Gm — — — — 56.1 22.8 32.3 59.2 PIP-72Gn — — — — —22.4 30.3 90.8 WP_028536646 — — — — — — 29.2 23.5 BTRA_1468 — — — — — —— 30.3 JG43047 — — — — — — — — PFL_6283 — — — — — — — — PIP-72Ba — — — —— — — — PIP-72Ca — — — — — — — — PIP-72Cb — — — — — — — — PIP-72Da — — —— — — — — PIP-72Db — — — — — — — — PIP-72Dc — — — — — — — — PIP-72Ea — —— — — — — — PIP-72Fa — — — — — — — — PIP-72Ff — — — — — — — — PIP-72Gb —— — — — — — — PIP-72Ge — — — — — — — — Plu2373 — — — — — — — —SRBS_294080 — — — — — — — — SwiRh_4910 — — — — — — — — XBJ1_1078 — — — —— — — — PFL_6283 PIP-72Ba PIP-72Ca PIP-72Cb PIP-72Da PIP-72Db PIP-72DcPIP-72Ea SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID NO: 30NO: 4 NO: 6 NO: 8 NO: 10 NO: 12 NO: 14 NO: 16 PIP-72Aa 34.8 83.7 72.169.8 68.6 69.8 69.8 51.7 (SEQ ID NO: 2) GBP_A3175 35.1 45.6 44.4 43.345.6 44.4 45.6 46.7 PIP-72Ab 32.6 82.6 69.8 67.4 66.3 67.4 67.4 52.9PIP-72Bb 35.9 91.9 68.6 70.9 67.4 68.6 68.6 52.9 WP_030131237 34.8 77.968.6 68.6 67.4 68.6 67.4 50.6 WP_016417435 35.9 51.7 47.7 46.5 47.7 46.547.7 43.7 BDL_2850 34.0 44.4 43.3 42.2 44.4 43.3 44.4 46.7 PIP-72Fh 47.340.7 35.2 37.4 36.3 37.4 36.3 35.2 PIP-72Fi 44.1 40.7 36.3 38.5 37.438.5 37.4 35.2 PIP-72Fj 48.0 33.3 35.4 34.4 36.5 35.4 36.5 35.4 PIP-72Fk43.9 35.4 34.4 33.3 35.4 34.4 35.4 35.4 PIP-72Fl 29.0 44.9 51.7 49.448.3 48.3 48.3 40.4 BBK_2354 35.1 44.4 43.3 42.2 44.4 43.3 44.4 46.7D512_15386 35.1 40.2 39.1 38.0 40.2 39.1 40.2 45.7 PIP-72Gg 69.5 35.831.6 32.6 32.3 31.2 32.3 34.4 PIP-72Gh 48.0 31.2 33.3 32.3 34.4 33.334.4 35.4 PIP-72Gi 46.2 38.5 34.1 36.3 35.2 36.3 35.2 33.0 BTI_1023 27.731.1 33.3 32.2 35.6 34.4 35.6 36.7 PIP-72Gk 47.3 40.7 39.6 41.8 40.739.6 40.7 33.7 PIP-72Gl 45.2 40.7 36.3 38.5 37.4 38.5 37.4 35.2 PIP-72Gm66.3 37.8 37.4 38.5 39.6 38.5 39.6 37.4 PIP-72Gn 46.0 31.6 33.7 32.734.7 33.7 34.7 34.7 WP_028536646 24.5 36.3 38.5 38.5 36.0 37.2 36.0 30.1BTRA_1468 28.7 32.2 34.4 33.3 35.6 34.4 35.6 37.8 JG43047 48.0 33.7 33.732.7 34.7 33.7 34.7 34.7 PFL_6283 — 35.9 33.7 34.8 34.8 33.7 34.8 35.9PIP-72Ba — — 72.1 74.4 69.8 70.9 70.9 51.7 PIP-72Ca — — — 97.7 93.0 93.091.9 46.0 PIP-72Cb — — — — 93.0 93.0 91.9 44.8 PIP-72Da — — — — — 97.798.8 44.8 PIP-72Db — — — — — — 97.7 46.0 PIP-72Dc — — — — — — — 44.8PIP-72Ea — — — — — — — — PIP-72Fa — — — — — — — — PIP-72Ff — — — — — — —— PIP-72Gb — — — — — — — — PIP-72Ge — — — — — — — — Plu2373 — — — — — —— — SRBS_294080 — — — — — — — — SwiRh_4910 — — — — — — — — X3J1_1078 — —— — — — — — PIP-72Fa PIP-72Ff PIP-72Gb PIP-72Ge Plu2373 SRBS_294080SwiRh_4910 XBJ1_1078 SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ ID SEQ IDSEQ ID NO: 18 NO: 28 NO: 32 NO: 38 NO: 36 NO: 22 NO: 26 NO: 34 PIP-72Aa38.5 36.7 34.4 33.7 38.9 41.1 38.9 36.7 (SEQ ID NO: 2) GBP_A3175 36.640.4 40.6 37.4 41.8 39.1 39.1 43.3 PIP-72Ab 38.5 34.7 32.3 31.6 39.438.9 36.7 35.6 PIP-72Bb 39.6 32.7 30.2 29.6 40.4 40.0 37.8 36.7WP_030131237 36.3 34.7 33.3 32.7 42.6 38.9 36.7 38.9 WP_016417435 31.936.6 34.3 33.7 31.2 37.6 35.5 36.7 BDL_2850 35.5 40.4 40.6 37.4 43.938.0 38.0 45.6 PIP-72Fh 95.6 54.1 53.1 52.0 24.0 56.0 57.1 31.5 PIP-72Fi97.8 55.1 54.2 53.1 25.3 56.0 57.1 30.4 PIP-72Fj 52.1 79.6 94.8 90.831.7 58.3 59.4 28.1 PIP-72Fk 55.2 96.9 78.1 80.6 32.7 54.2 54.2 28.1PIP-72Fl 27.2 32.3 35.1 35.4 32.3 34.1 35.9 31.5 BBK_2354 35.5 40.4 40.637.4 42.9 39.1 39.1 44.4 D512_15386 34.7 38.6 40.8 37.6 38.0 36.2 36.240.2 PIP-72Gg 41.7 42.7 49.5 49.5 27.9 63.2 67.4 28.4 PIP-72Gh 52.1 79.692.7 95.9 30.8 58.3 59.4 28.1 PIP-72Gi 96.7 57.1 56.2 55.1 24.0 58.259.3 29.3 BTI_1023 31.2 32.3 32.3 29.3 36.1 30.4 30.4 37.5 PIP-72Gk 52.252.0 49.5 50.0 33.3 50.5 51.6 31.9 PIP-72Gl 98.9 54.1 53.1 52.0 24.056.0 57.1 30.4 PIP-72Gm 56.0 53.1 59.4 58.2 31.3 93.3 98.9 31.9 PIP-72Gn53.1 79.6 92.9 94.9 30.2 55.1 56.1 27.6 WP_028536646 24.2 21.4 24.0 23.534.7 23.3 22.8 37.1 BTRA_1468 32.3 34.7 30.9 30.3 37.1 32.3 32.3 38.6JG43047 52.0 82.7 91.8 91.8 32.1 58.2 59.2 27.6 PFL_6283 45.2 44.0 48.047.0 22.9 63.0 67.4 29.0 PIP-72Ba 39.6 34.7 32.3 31.6 41.5 41.1 38.935.6 PIP-72Ca 35.2 33.7 34.4 33.7 40.4 35.6 37.4 37.8 PIP-72Cb 37.4 32.733.3 32.7 40.4 36.7 38.5 37.8 PIP-72Da 36.3 34.7 35.4 34.7 41.5 37.839.6 34.4 PIP-72Db 37.4 33.7 34.4 33.7 39.4 36.7 38.5 35.6 PIP-72Dc 36.334.7 35.4 34.7 41.5 37.8 39.6 34.4 PIP-72Ea 34.1 34.7 35.4 34.7 35.835.6 37.4 35.6 PIP-72Fa — 54.1 53.1 52.0 24.0 56.0 57.1 30.4 PIP-72Ff —— 77.6 83.7 32.1 54.1 54.1 27.6 PIP-72Gb — — — 92.9 30.8 58.3 59.4 28.1PIP-72Ge — — — — 30.2 57.1 58.2 27.6 Plu2373 — — — — — 31.6 31.3 41.2SRBS_294080 — — — — — — 94.4 33.0 SwiRh_4910 — — — — — — — 31.9X3J1_1078 — — — — — — — —

Example 3—E. coli Expression of PIP-72Aa and Homologs

The PIP-72Aa gene was amplified by PCR using genomic DNA isolated fromstrain SS143D5: forward primer (SEQ ID NO: 39) and reverse primer (SEQID NO: 40). The resulting PCR product was DNA sequence verified andsubcloned into pCOLD™ 1 (Takara Bio Inc., Seta 3-4-1, Otsu, Shiga, Japan520-2193) in frame with an N-terminal His-6 tag followed by a Factor Xacleavage site. The polynucleotides encoding PIP-72Aa homologs identifiedfrom internal strains (PIP-72Ba, PIP-72Ca, PIP-72Cb, PIP-72 Da,PIP-72Db, PIP-72Dc, PIP-72Db, PIP-72Ea, PIP-72Fa, JG43047, IDP072Ff,PFL_6283, PIP-72Gb, PIP-72Ge) were cloned as described above, usingtheir respective genomic DNA preparation as template for geneamplification by PCR and the primer sequences indicated in Table 8.

Homologs of PIP-72Aa which were identified from external databases(GBP_A3175, SRBS_294080, SwiRh_4910, XBJ1_1078, Plu2373, WP_030131237,and WP_016417435) were obtained through gene synthesis with compatible5′ and 3′ ends for downstream cloning into pCOLD™-1.

TABLE 8 gene forward primer reverse primer PIP-72Ba SEQ ID NO: 41 SEQ IDNO: 42 PIP-72Ca SEQ ID NO: 43 SEQ ID NO: 44 PIP-72Cb SEQ ID NO: 45 SEQID NO: 46 PIP-72Da SEQ ID NO: 47 SEQ ID NO: 48 PIP-72Dc SEQ ID NO: 49SEQ ID NO: 50 PIP-72Db SEQ ID NO: 51 SEQ ID NO: 52 PIP-72Ea SEQ ID NO:53 SEQ ID NO: 54 PIP-72Fa SEQ ID NO: 55 SEQ ID NO: 56 JG43047 SEQ ID NO:57 SEQ ID NO: 58 PIP-72Ff SEQ ID NO: 59 SEQ ID NO: 60 PFL_6283 SEQ IDNO: 61 SEQ ID NO: 62 PIP-72Gb SEQ ID NO: 63 SEQ ID NO: 64 PIP-72Ge SEQID NO: 65 SEQ ID NO: 66 PIP-72Ge SEQ ID NO: 67 SEQ ID NO: 68 PIP-72Ab SEQ ID NO: 971  SEQ ID NO: 972 PIP-72Bb  SEQ ID NO: 973  SEQ ID NO: 974PIP-72Fi  SEQ ID NO: 975  SEQ ID NO: 976 PIP-72Gh  SEQ ID NO: 977  SEQID NO: 978 PIP-72Gg  SEQ ID NO: 979  SEQ ID NO: 980

Competent BL21-DE3 E. coli cells were transformed with pCOLD™-1 plasmidDNA, containing the respective PIP-72 gene insert for recombinantprotein expression. The transformed E. coli cells were grown overnightat 37° C. with carbenicillin selection and then inoculated to a fresh2×YT medium (1:25) and further grown to an optical density of about 0.8.The cells were then chilled in the presence of 1 mM ITPG and furthergrown at 16° C. for 16 hours to induce protein expression. The E. coliexpressed proteins were purified by immobilized metal ion chromatographyusing Ni-NTA agarose (Qiagen®, Germany) according to the manufacturer'sprotocols.

Example 4—Insect Activity of PIP-72Aa and Homologs

A series of concentrations of the purified protein sample was assayedagainst Coleoptera insects and concentrations for 50% mortality (LC50)or inhibition of 50% of the individuals (IC50) were calculated in twoindependent experiments. The WCRW results for PIP-72Aa (SEQ ID NO: 2)are shown in Table 9.

TABLE 9 ppm 95% confidence interval (ppm) LC50 80 30-130 IC50 20 10-40 

To measure insecticidal activities of other PIP-72 polypeptides, WCRW(Diabrotica virgifera virgifera) bioassays were conducted using 20 ul ofthe purified protein samples applied topically over 75 ul artificialWCRW diet (Bio-Serv F9800B based) in each of a 96 well bioassay plate(BD Falcon 353910) then air dried. A variable number of neonateDiabrotica virgifera virgifera neonates (3 to 9) were placed into eachwell of the 96 well plate. The assay was run for 4 days at 25° C. withno light and then scored for mortality and stunting. The WCRWinsecticidal activity for the various PIP-72 polypeptides is shown inTable 10.

PIP-72Aa (SEQ ID NO: 2) was further tested against Southern Corn RootWorm (Diabrotica undecimpunctata howardi) and San Antonio beetle(Diabrotica speciosa) as well as against the sucking insect Lygushesperus. PIP-72Aa (SEQ ID NO: 2) was not insecticidal against thesepests at the concentrations tested (up to 875 ppm of purified protein).

Several PIP-72 polypeptides were also tested against SCRW (Diabroticaundecimpunctata howardi). Bioassays were conducted using 10 ul of thepurified protein samples mixed with 50 ul artificial SCRW diet (Bio-ServF9800B based) in each of a 96 well bioassay plate (BD Falcon 353910). Avariable number of neonate Diabrotica undecimpunctata howardi neonates(3 to 5) were placed into each well of the 96 well plate. The assay wasrun for 4 days at 25° C. with no light and then scored for mortality andstunting.

TABLE 10 WCRW highest conc. activity, tested, ug/cm2 IC50, IC50, effect(ppm) ug/cm2 ppm PIP-72Aa active, death 300  5-15 10-40 SEQ ID NO: 2PIP-72Ab active, death 1000 50 SEQ ID NO: 927 PIP-72Ba active, death(1900) 10-40 SEQ ID NO: 4 PIP-72Bb active, death 1000 50 SEQ ID NO: 928PIP-72Ca active, death 1500 10-30 70 SEQ ID NO: 6 PIP-72Cb active, death1000 10-30 84 SEQ ID NO: 8 PIP-72Da active, death (1666) not tested in40-80 SEQ ID NO: 10 overlay format PIP-72Db active, death 750 10-30 95SEQ ID NO: 12 PIP-72Dc active, death 450  5-15 ~100 SEQ ID NO: 14PIP-72Ea Inactive at (2150) SEQ ID NO: 16 concentration tested PIP-72Faactive, 1132 141 SEQ ID NO: 18 severe stunting PIP-72Fi active, 1000 SEQID NO: 933 severe stunting PIP-72Gg active, 1000 500 SEQ ID NO: 939severe stunting PIP-72Gh active, 1000 SEQ ID NO: 940 severe stuntingGBP_A3175 active, death 224 15-30 SEQ ID NO: 20 SRBS_294080 notexpressed — — SEQ ID NO: 22 JG43047 weakly 218 >218 SEQ ID NO: 24active, mild stunting SwiRh_4910 Inactive at 708 — SEQ ID NO: 26concentration tested WP_030131237 active, death 400 SEQ ID NO: 929WP_016417435 inactive, low SEQ ID NO: 930 expression PIP-72Ff weakly 565~500 SEQ ID NO: 28 active, stunting PFL_6283 weakly 414 >414 SEQ ID NO:30 active, mild stunting PIP-72Gb active, 571 71 SEQ ID NO: 32 severestunting XBJ1_1078 inactive 97 — SEQ ID NO: 34 Plu2373 not expressed — —SEQ ID NO: 36 PIP-72Ge inactive 457 — SEQ ID NO: 38

Lepidoptera feeding assays were conducted on an artificial diet in a 96well plate set up. The purified protein was incorporated with theLepidopteran-specific artificial diet in a ratio of 10 ul cleared lysateand 40 ul of diet mixture. Two to five neonate larvas were placed ineach well to feed ad libitum for 5 days. Results were expressed aspositive for larvae reactions such as stunting and or mortality. Resultswere expressed as negative if the larvae were similar to the negativecontrol that is feeding diet to which the above buffer only has beenapplied.

PIP-72Aa (SEQ ID NO: 2), PFL_6283 (SEQ ID NO: 30), PIP-72 Da (SEQ ID NO:10), PIP-72Fa (SEQ ID NO: 18) and PIP-72Gb (SEQ ID NO: 32) were assayedon European corn borer (Ostrinia nubilalis), corn earworm (Helicoverpazea), black cutworm (Agrotis ipsilon), fall armyworm (Spodopterafrugiperda) and Soybean looper (Pseudoplusia includens). No activityagainst the Lepidoptera species was seen for any of the PIP-72polypeptides tested at protein concentrations up to 800 ppm.

Example 5—Lack of Cross Resistance of PIP-72Aa in mCry3A ResistantStrain of WCRW

The WCRW strain resistant to mCry3A was developed by selections of WCRWon mCry3A transgenic maize plants with T0 expression level of mCry3Aat >10,000 ppm of total proteins in roots. Seven selections were made onF3, F6, F7, F8, F10, F12, F14 larvae. F16 eggs of the Cry3A-resistantinsects had a resistance ratio (RR) of >46-fold to mCry3A compared withthe susceptible laboratory colony, and were used for cross resistancetesting of PIP-72Aa (SEQ ID NO: 2). Standardized WCRW diet incorporationbioassays were utilized to evaluate the effects of PIP-72Aa (SEQ ID NO:2) on WCRW larvae. WCRW neonate larvae were placed on the platescontaining the bioassay diet and insecticidal protein with 4 replicatesfor each concentration treatment for 3 days after initiation of eachbioassay. Insect mortality and severe stunting was scored and used tocalculate inhibitory concentrations (IC50 and LC50) based on probitanalysis. The resistance ratio (RR) was calculated as follows:RR=(LC/IC50 of resistant WCRW)/(LC/IC50 of susceptible WCRW). As shownin Table 11 Cry3A-resistant WCRW insects were sensitive to PIP-72Aa (SEQID NO: 2).

TABLE 11 PIP-72Aa (SEQ ID NO: 2), Resistance WCRW colony LC/IC ppm 95%CL Ratio Cry3A sensitive LC50 168.9 97.81-429.5 1 IC50 15.38 11.64-19.681 Cry3A resistant LC50 212.3 102.9-1071  1.3 IC50 25.97 17.71-38.35 1.7

Example 6—Agrobacterium-Mediated Stable Transformation of Maize

For Agrobacterium-mediated maize transformation PIP-72 polypeptides, themethod of Zhao was employed (U.S. Pat. No. 5,981,840 and InternationalPatent Publication Number WO 1998/32326, the contents of which arehereby incorporated by reference). Briefly, immature embryos wereisolated from maize and the embryos contacted with an AgrobacteriumSuspension, where the bacteria were capable of transferring apolynucleotide encoding a PIP-72 polypeptide to at least one cell of atleast one of the immature embryos (step 1: the infection step). In thisstep the immature embryos were immersed in an Agrobacterium suspensionfor the initiation of inoculation. The embryos were co-cultured for atime with the Agrobacterium (step 2: the co-cultivation step). Theimmature embryos were cultured on solid medium with antibiotic, butwithout a selecting agent, for Agrobacterium elimination and for aresting phase for the infected cells. Next, inoculated embryos werecultured on medium containing a selective agent and growing transformedcallus is recovered (step 4: the selection step). The immature embryoswere cultured on solid medium with a selective agent resulting in theselective growth of transformed cells. The callus was then regeneratedinto plants (step 5: the regeneration step), and calli grown onselective medium were cultured on solid medium to regenerate the plants.

For detection of the PIP-72 polypeptide in leaf tissue 4 lyophilizedleaf punches/sample were pulverized and resuspended in 100 μL PBScontaining 0.1% TWEEN™ 20 (PBST), 1% beta-mercaoptoethanol containing 1tablet/7 mL complete Mini proteinase inhibitor (Roche 1183615301). Thesuspension was sonicated for 2 min and then centrifuged at 4°, 20,000 gfor 15 min. To a supernatant aliquot ⅓ volume of 3× NuPAGE® LDS SampleBuffer (Invitrogen™ (CA, USA), 1% B-ME containing 1 tablet/7 mL completeMini proteinase inhibitor was added. The reaction was heated at 80° C.for 10 min and then centrifuged. A supernatant sample was loaded on4-12% Bis-Tris Midi gels with MES running buffer as per manufacturer's(Invitrogen™) instructions and transferred onto a nitrocellulosemembrane using an iBlot® apparatus (Invitrogen™). The nitrocellulosemembrane was incubated in PBST containing 5% skim milk powder for 2hours before overnight incubation in affinity-purified rabbitanti-PIP-72Aa in PBST overnight. The membrane was rinsed three timeswith PBST and then incubated in PBST for 15 min and then two times 5 minbefore incubating for 2 hours in PBST with goat anti-rabbit-HRP for 3hours. The detected proteins were visualized using ECL Western BlottingReagents (GE Healthcare cat # RPN2106) and Kodak® Biomax® MR film. Fordetection of the PIP-72Aa protein in roots the roots were lyophilizedand 2 mg powder per sample was resuspended in LDS, 1%beta-mercaptoethanol containing 1 tablet/7 mL Complete Mini proteinaseinhibitor was added. The reaction was heated at 80° C. for 10 min andthen centrifuged at 4° C., 20,000 g for 15 min. A supernatant sample wasloaded on 4-12% Bis-Tris Midi gels with MES running buffer as permanufacturer's (Invitrogen™) instructions and transferred onto anitrocellulose membrane using an iBlot® apparatus (Invitrogen™). Thenitrocellulose membrane was incubated in PBST containing 5% skim milkpowder for 2 hours before overnight incubation in affinity-purifiedpolyclonal rabbit anti-PIP-72Aa antibody in PBST overnight. The membranewas rinsed three times with PBST and then incubated in PBST for 15 minand then two times 5 min before incubating for 2 hours in PBST with goatanti-rabbit-HRP for 3 hrs. The antibody bound insecticidal proteins weredetected using ECL™ Western Blotting Reagents (GE Healthcare cat#RPN2106) and Kodak® Biomax® MR film.

Transgenic maize plants positive for expression of the insecticidalproteins are tested for pesticidal activity using standard bioassaysknown in the art. Such methods include, for example, root excisionbioassays and whole plant bioassays. See, e.g., US Patent ApplicationPublication Number US 2003/0120054 and International Publication NumberWO 2003/018810.

Example 7—Expression Vector Constructs for Expression of PIP-72Aa inPlants

The plant expression vectors, PHP61666, PHP61668, PHP61664 wereconstructed to include a transgene cassette containing the nativePIP-72Aa (SEQ ID NO: 1), PIP-72Aa maize optimized variant 1 (SEQ ID NO:850) and PIP-72Aa maize optimized variant 2 (SEQ ID NO: 851),respectively, under control of the Mirabilis Mosaic Virus (MMV) promoter[Dey N and Maiti I B, 1999, Plant Mol. Biol. 40(5):771-82] incombination with an enhancer element. Additional vectors, PHP64465,PHP64471, and PHP64468 expressing the PIP-72Aa (maize optimizedVariant 1) under different promoter, intron and terminator combinationswere also tested in transgenic maize events. PHP64465 expresses PIP-72Aa(Variant 1) under control of the maize polyubiquitin promoter andassociated 5′UTR and intron (Christensen A H and Quail R H, 1996,Transgenic Res 5:213-218) combined with the 35S enhancer (Kay et al.,1987, Science 236(4806):1299-1302. PHP64471 and PHP64468 express Variant1 under control of the Banana Streak Virus [BSV(AY)TR] promoter (DiehnS, Lu A L and Simmons C R, 2012, U.S. Pat. No. 8,338,662B2) with eitherthe ZM-HPLV9 (Diehn S et al., 2011, US2011039696) or ZM-ADH1 intron(Callis et al, 1987, Genes Develop 1:1183-1200), respectively. PHP69828expresses PIP-72Aa maize optimized variant 3 (SEQ ID NO: 853) with thesame regulatory elements as in PHP64471.

These constructs were used to generate transgenic maize events to testfor efficacy against corn rootworm provided by expression of PIP-72Aa(SEQ ID NO: 2).

T0 greenhouse efficacy results for events generated from theseconstructs are shown in FIG. 8. Efficacy for events derived from each ofthe constructs was observed relative to negative control events asmeasured by root protection from Western corn root worm. Root protectionwas measured according to the number of nodes of roots injured(CRWNIS=corn rootworm node injury score) using the method developed byOleson, et al. (2005) [J. Econ Entomol. 98(1):1-8]. The root injuryscore is measured from “0” to “3” with “0” indicating no visible rootinjury, “1” indicating 1 node of root damage, “2” indicating 2 nodes orroot damage, and “3” indicating a maximum score of 3 nodes of rootdamage. Intermediate scores (e.g. 1.5) indicate additional fractions ofnodes of damage (e.g. one and a half nodes injured).

FIG. 8 shows that the majority of events from the test constructs (eachrepresents a single event from the construct) performed better than thenegative control with rootworm injury scores of <1.0. Several constructssuch as PHP64471 and PHP64468 showed rootworm injury scores thataveraged <0.2 across all events tested.

Example 8—PIP-72Aa Variants with Multiple Amino Acid Substitutions

To create variants of PIP-72Aa (SEQ ID NO: 2) with multiple amino acidchanges, variant libraries were generated by synthetic DNA shuffling(Ness et al, 2002, Nature Biotechnology 20, 1251-5) of PIP-72Aa (SEQ IDNO: 1) and GBP_A3175 (SEQ ID NO: 19) or by site-directed mutagenesis(QuikChange™ Lightning technique or QuikChange™ II technique, Agilent,Santa Clara Calif.). An amino acid sequence alignment of PIP-72Aa (SEQID NO: 2) and GBP_A3175 (SEQ ID NO: 20) is shown in FIG. 5. Briefly,gene synthesis reactions were performed with oligonucleotides listed inTable 12.

TABLE 12 Name Sequence oligo 10 SEQ ID NO: 69 oligo 103 SEQ ID NO: 70oligo 104 SEQ ID NO: 71 oligo 105 SEQ ID NO: 72 oligo 106 SEQ ID NO: 73oligo 107 SEQ ID NO: 74 oligo 108 SEQ ID NO: 75 oligo 109 SEQ ID NO: 76oligo 110 SEQ ID NO: 77 oligo 111 SEQ ID NO: 78 oligo 112 SEQ ID NO: 79oligo 113 SEQ ID NO: 80 oligo 114 SEQ ID NO: 81 oligo 115 SEQ ID NO: 82oligo 116 SEQ ID NO: 83 oligo 117 SEQ ID NO: 84 oligo 118 SEQ ID NO: 85oligo 119 SEQ ID NO: 86 oligo 120 SEQ ID NO: 87 oligo 121 SEQ ID NO: 88oligo 122 SEQ ID NO: 89 oligo 123 SEQ ID NO: 90 oligo 124 SEQ ID NO: 91oligo 125 SEQ ID NO: 92 oligo 126 SEQ ID NO: 93 oligo 127 SEQ ID NO: 94oligo 128 SEQ ID NO: 95 oligo 129 SEQ ID NO: 96 oligo 130 SEQ ID NO: 97oligo 131 SEQ ID NO: 98 oligo 132 SEQ ID NO: 99 oligo 133 SEQ ID NO: 100oligo 134 SEQ ID NO: 101 oligo 135 SEQ ID NO: 102 oligo 136 SEQ ID NO:103 oligo 137 SEQ ID NO: 104 oligo 138 SEQ ID NO: 105 oligo 139 SEQ IDNO: 106 oligo 140 SEQ ID NO: 107 oligo 141 SEQ ID NO: 108 oligo 142 SEQID NO: 109 oligo 143 SEQ ID NO: 110 oligo 144 SEQ ID NO: 111 oligo 145SEQ ID NO: 112 oligo 146 SEQ ID NO: 113 oligo 147 SEQ ID NO: 114 oligo148 SEQ ID NO: 115 oligo 149 SEQ ID NO: 116 oligo 150 SEQ ID NO: 117oligo 151 SEQ ID NO: 118 oligo 152 SEQ ID NO: 119 oligo 153 SEQ ID NO:120 oligo 154 SEQ ID NO: 121 oligo 155 SEQ ID NO: 122 oligo 156 SEQ IDNO: 123 oligo 157 SEQ ID NO: 124 oligo 158 SEQ ID NO: 125 oligo 159 SEQID NO: 126 oligo 160 SEQ ID NO: 127 oligo 161 SEQ ID NO: 128 oligo 162SEQ ID NO: 129 oligo 163 SEQ ID NO: 130 oligo 164 SEQ ID NO: 131 oligo165 SEQ ID NO: 132 oligo 166 SEQ ID NO: 133 oligo 167 SEQ ID NO: 134oligo 168 SEQ ID NO: 135 oligo 169 SEQ ID NO: 136 oligo 170 SEQ ID NO:137 oligo 171 SEQ ID NO: 138 oligo 172 SEQ ID NO: 139 oligo 173 SEQ IDNO: 140 oligo 174 SEQ ID NO: 141 oligo 175 SEQ ID NO: 142

Several synthetic genes were generated using different primer sets asindicated in Table 13. Gene synthesis reactions F46, F31 and F39 eachutilized sets of 10 discrete oligonucleotide primers. Gene synthesisreactions deg7, deg15, deg20 utilized sets of oligonucleotide primerswith sequence degeneracy, resulting in libraries of sequences. Genesynthesis reactions F46, F31, F39, deg7, deg15 and deg20 were combinedand screened as a single library.

TABLE 13 Reaction Primers F46 10, 103, 104, 105, 106, 107, 108, 109,110, 111 deg7 10, 112, 113, 114, 115, 116, 117, 118, 119, 120 deg15 10,121, 122, 123, 124, 125, 126, 127, 128, 129 F31 10, 130, 131, 132, 133,134, 135, 136, 137, 138 deg20 10, 139,140, 141, 142, 143, 144, 145, 146,147, 148, 149, 150, 151, 152, 153, 154, 155, 156, 157, 158, 159, 160,161, 162, 163, 164, 165, 166 F39 10, 167, 168, 169, 170, 171, 172, 173,174, 175

Synthetic gene fragments were subcloned in expression vector pCOLD™1 asdescribed in Example 3. The resulting plasmid DNAs were transformed intoE. coli, and protein was expressed as described in Example 3. Bioassayswere performed on Western Corn Root Worm as described in Example 4 todetermine which gene variants retained insecticidal activity. DNAsequencing was performed on the active gene variants. The nucleotide andamino acid sequences of the resulting active PIP-72 polypeptide variantsare listed in Table 14. Five active variants were recovered from 59unique variants that were screened. Table 15 shows the amino acidsubstitutions of the resulting active PIP-72 polypeptide variantscompared to PIP-72Aa (SEQ ID NO: 2).

TABLE 14 % Identity to PIP72Aa Clone Polynucleotide Polypeptide SEQ IDNO: 2 designation SEQ ID NO SEQ ID NO 53% PIP-72Aa-Fb-01 SEQ ID NO: 287SEQ ID NO: 528 48% PIP-72Aa-Fb-09 SEQ ID NO: 288 SEQ ID NO: 529 56%PIP-72Aa-Fb-27 SEQ ID NO: 289 SEQ ID NO: 530 57% PIP-72Aa-Fb-28 SEQ IDNO: 290 SEQ ID NO: 531 67% PIP-72Aa-Fb-33 SEQ ID NO: 291 SEQ ID NO: 532

TABLE 15 Clone Amino acid substitutions compared to DesignationPolypeptide PIP-72Aa (SEQ ID NO: 2) PIP-72Aa-Fb-01 SEQ ID NO: G002A,T004S, T006K, S009A, I013V, 528 S022T, S027K, F028P, G032A, N033P,K035A, Q036S, E037D, T038S, S042N, S044L, L049M, S050Y, L051V, K052Q,K053L, N054G, A056S, Q057A, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LPIP-72Aa-Fb-09 SEQ ID NO: G002A, T004S, T006K, N008S, S009A, 529 P012T,I013V, A016S, S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, E037D, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L, N054G,A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LPIP-72Aa-Fb-27 SEQ ID NO: G002A, T006K, N008S, S009A, P012T, 530 A016S,S022T, S027K, F028P, S030K, G032A, N033P, K035A, Q036S, S042N, S044L,S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S,K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q,L081T, E083L PIP-72Aa-Fb-28 SEQ ID NO: G002A, T006K, N008S, S009A,P012T, 531 A016S, S027K, F028P, S030K, G032A, N033P, K035A, Q036S,S042N, S044L, S050Y, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L,A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T,Q078S, R080Q, L081T, E083L PIP-72Aa-Fb-33 SEQ ID NO: G002A, T004S,S009A, I013V, S022T, 532 G032A, E037D, T038S, S044L, L049M, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L, E083L

To generate additional active PIP-72 polypeptides, further site-directedmutagenesis was performed using the polynucleotide sequencesPIP-72Aa-Fb-9 (SEQ ID NO: 288), PIP-72Aa-Fb-27 (SEQ ID NO: 289) andPIP-72Aa-Fb-33 (SEQ ID NO: 291) as the templates by the QuikChange™technique (Agilent, 5301 Stevens Creek Blvd., Santa Clara Calif.) usingoligonucleotide primers listed in Table 16. Primers PIP-72Aa-Fb_9-27_1(SEQ ID NO: 143), PIP-72Aa-Fb_9-27_2 (SEQ ID NO: 144),PIP-72Aa-Fb_9-27_3 (SEQ ID NO: 145), PIP-72Aa-Fb_9-27_12 (SEQ ID NO:146), PIP-72Aa-Fb_9-27_13 (SEQ ID NO: 147), and PIP-72Aa-Fb_9-27_14 (SEQID NO: 148) were used for mutagenesis of plasmid DNA templatesPIP-72Aa-Fb-09 (SEQ ID NO: 288) and PIP-72Aa-Fb-27 (SEQ ID NO: 289).Primers PIP-72Aa-Fb_33_1 through PIP-72Aa-Fb_33_11 (SEQ ID NO: 149through SEQ ID NO: 159) were used for mutagenesis of plasmid DNAtemplate PIP-72Aa-Fb-33 (SEQ ID NO: 291).

TABLE 16 Sequence Primer Name identifier PIP-72Aa-Fb_9-27_1 SEQ ID NO:143 PIP-72Aa-Fb_9-27_2 SEQ ID NO: 144 PIP-72Aa-Fb_9-27_3 SEQ ID NO: 145PIP-72Aa-Fb_9-27_12 SEQ ID NO: 146 PIP-72Aa-Fb_9-27_13 SEQ ID NO: 147PIP-72Aa-Fb_9-27_14 SEQ ID NO: 148 PIP-72Aa-Fb_33_1 SEQ ID NO: 149PIP-72Aa-Fb_33_2 SEQ ID NO: 150 PIP-72Aa-Fb_33_3 SEQ ID NO: 151PIP-72Aa-Fb_33_4 SEQ ID NO: 152 PIP-72Aa-Fb_33_5 SEQ ID NO: 153PIP-72Aa-Fb_33_6 SEQ ID NO: 154 PIP-72Aa-Fb_33_7 SEQ ID NO: 155PIP-72Aa-Fb_33_8 SEQ ID NO: 156 PIP-72Aa-Fb_33_9 SEQ ID NO: 157PIP-72Aa-Fb_33_10 SEQ ID NO: 158 PIP-72Aa-Fb_33_11 SEQ ID NO: 159PIP-72Aa-Fb_33_12 SEQ ID NO: 160 PIP-72Aa-Fb_33_13 SEQ ID NO: 161PIP-72Aa-Fb_33_14 SEQ ID NO: 162 PIP-72Aa-Fb_33_15 SEQ ID NO: 163PIP-72Aa-Fb_33_16 SEQ ID NO: 164 PIP-72Aa-Fb_33_17 SEQ ID NO: 165PIP-72Aa-Fb_33_18 SEQ ID NO: 166 PIP-72Aa-Fb_33_19 SEQ ID NO: 167PIP-72Aa-Fb_33_20 SEQ ID NO: 168 PIP-72Aa-Fb_33_21 SEQ ID NO: 169PIP-72Aa-Fb_33_22 SEQ ID NO: 170 PIP-72Aa-Fb_33_9 SEQ ID NO: 157PIP-72Aa-Fb_33_10 SEQ ID NO: 158

The resulting plasmid DNAs were transformed into E. coli, and proteinwas expressed as described in Example 3. Bioassays were performed onWestern Corn Root Worm as described in Example 4 to determine which genevariants retained insecticidal activity. DNA sequencing was performed onthe active gene variants. The percent identity compared to PIP-72Aa (SEQID NO: 2), clone identification, polynucleotide and polypeptidesequences of the resulting active variants are listed in Table 17. ThePIP-72 polypeptide variants from PIP-72Aa-Fb-9 (SEQ ID NO: 530) yielded33 active variants out of 72 that were screened. The PIP-72 polypeptidevariants from PIP-72Aa-Fb-27 (SEQ ID NO: 530) yielded 11 active variantsout of 52 that were screened. PIP-72 polypeptide variants fromPIP-72Aa-Fb-33 (SEQ ID NO: 532) yielded 47 active variants out of 79that were screened. Table 18 shows the amino acid substitutions of theresulting active PIP-72 polypeptide variants compared to PIP-72Aa (SEQID NO: 2).

TABLE 17 % Identity to Clone Polynucleotide Polypeptide PIP-72Aadesignation SEQ ID NO SEQ ID NO: 55% D_D0414317 SEQ ID NO: 292 SEQ IDNO: 533 56% D_D0414326 SEQ ID NO: 293 SEQ ID NO: 534 57% D_D0414327 SEQID NO: 294 SEQ ID NO: 535 55% D_D0414355 SEQ ID NO: 295 SEQ ID NO: 53653% D_D0414366 SEQ ID NO: 296 SEQ ID NO: 537 57% D_D0414388 SEQ ID NO:297 SEQ ID NO: 538 52% D_D0414411 SEQ ID NO: 298 SEQ ID NO: 539 53%D_D0414412 SEQ ID NO: 299 SEQ ID NO: 540 49% D_D0414416 SEQ ID NO: 300SEQ ID NO: 541 48% D_D0414422 SEQ ID NO: 301 SEQ ID NO: 542 50%D_D0414424 SEQ ID NO: 302 SEQ ID NO: 543 49% D_D0414425 SEQ ID NO: 303SEQ ID NO: 544 53% D_D0414427 SEQ ID NO: 304 SEQ ID NO: 545 46%D_D0414430 SEQ ID NO: 305 SEQ ID NO: 546 51% D_D0414433 SEQ ID NO: 306SEQ ID NO: 547 50% D_D0414439 SEQ ID NO: 307 SEQ ID NO: 548 49%D_D0414449 SEQ ID NO: 308 SEQ ID NO: 549 49% D_D0414450 SEQ ID NO: 309SEQ ID NO: 550 49% D_D0414453 SEQ ID NO: 310 SEQ ID NO: 551 48%D_D0414454 SEQ ID NO: 311 SEQ ID NO: 552 52% D_D0414455 SEQ ID NO: 312SEQ ID NO: 553 50% D_D0414459 SEQ ID NO: 313 SEQ ID NO: 554 48%D_D0414462 SEQ ID NO: 314 SEQ ID NO: 555 50% D_D0414466 SEQ ID NO: 315SEQ ID NO: 556 51% D_D0414470 SEQ ID NO: 316 SEQ ID NO: 557 49%D_D0414471 SEQ ID NO: 317 SEQ ID NO: 558 51% D_D0414475 SEQ ID NO: 318SEQ ID NO: 559 50% D_D0414479 SEQ ID NO: 319 SEQ ID NO: 560 51%D_D0414486 SEQ ID NO: 320 SEQ ID NO: 561 50% D_D0414489 SEQ ID NO: 321SEQ ID NO: 562 50% D_D0414493 SEQ ID NO: 322 SEQ ID NO: 563 69%D_D0419050 SEQ ID NO: 323 SEQ ID NO: 564 70% D_D0419051 SEQ ID NO: 324SEQ ID NO: 565 70% D_D0419053 SEQ ID NO: 325 SEQ ID NO: 566 69%D_D0419057 SEQ ID NO: 326 SEQ ID NO: 567 71% D_D0419058 SEQ ID NO: 327SEQ ID NO: 568 66% D_D0419060 SEQ ID NO: 328 SEQ ID NO: 569 71%D_D0419062 SEQ ID NO: 329 SEQ ID NO: 570 70% D_D0419064 SEQ ID NO: 330SEQ ID NO: 571 66% D_D0419066 SEQ ID NO: 331 SEQ ID NO: 572 69%D_D0419071 SEQ ID NO: 332 SEQ ID NO: 573 70% D_D0419072 SEQ ID NO: 333SEQ ID NO: 574 69% D_D0419075 SEQ ID NO: 334 SEQ ID NO: 575 72%D_D0419077 SEQ ID NO: 335 SEQ ID NO: 576 71% D_D0419082 SEQ ID NO: 336SEQ ID NO: 577 69% D_D0419083 SEQ ID NO: 337 SEQ ID NO: 578 66%D_D0419084 SEQ ID NO: 338 SEQ ID NO: 579 70% D_D0419087 SEQ ID NO: 339SEQ ID NO: 580 69% D_D0419091 SEQ ID NO: 340 SEQ ID NO: 581 73%D_D0419093 SEQ ID NO: 341 SEQ ID NO: 582 70% D_D0419095 SEQ ID NO: 342SEQ ID NO: 583 71% D_D0419096 SEQ ID NO: 343 SEQ ID NO: 584 70%D_D0419098 SEQ ID NO: 344 SEQ ID NO: 585 70% D_D0419099 SEQ ID NO: 345SEQ ID NO: 586 70% D_D0419101 SEQ ID NO: 346 SEQ ID NO: 587 69%D_D0419102 SEQ ID NO: 347 SEQ ID NO: 588 71% D_D0419106 SEQ ID NO: 348SEQ ID NO: 589 70% D_D0419108 SEQ ID NO: 349 SEQ ID NO: 590 67%D_D0419109 SEQ ID NO: 350 SEQ ID NO: 591 67% D_D0419110 SEQ ID NO: 351SEQ ID NO: 592 69% D_D0419111 SEQ ID NO: 352 SEQ ID NO: 593 67%D_D0419114 SEQ ID NO: 353 SEQ ID NO: 594 70% D_D0419115 SEQ ID NO: 354SEQ ID NO: 595 72% D_D0419117 SEQ ID NO: 355 SEQ ID NO: 596 74%D_D0419118 SEQ ID NO: 356 SEQ ID NO: 597 67% D_D0419119 SEQ ID NO: 357SEQ ID NO: 598 69% D_D0419120 SEQ ID NO: 358 SEQ ID NO: 599 72%D_D0419124 SEQ ID NO: 359 SEQ ID NO: 600 72% D_D0419126 SEQ ID NO: 360SEQ ID NO: 601 71% D_D0419127 SEQ ID NO: 361 SEQ ID NO: 602 70%D_D0419128 SEQ ID NO: 362 SEQ ID NO: 603 71% D_D0419132 SEQ ID NO: 363SEQ ID NO: 604 69% D_D0419138 SEQ ID NO: 364 SEQ ID NO: 605 65%D_D0419142 SEQ ID NO: 365 SEQ ID NO: 606 69% D_D0419145 SEQ ID NO: 366SEQ ID NO: 607

TABLE 18 Clone Amino acid substitutions compared to DesignationPolypeptide PIP-72Aa (SEQ ID NO: 2) D_D0414317 SEQ ID NO: G002A, T004S,T006K, N008S, S009A, 533 P012T, I013V, S022T, S027K, F028P, S030K,G032A, N033P, K035A, Q036S, S042N, S044L, S050Y, L051V, K052Q, K053L,N054G, A056S, Q057A, H058T, Q063L, A064S, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, E083L D_D0414326 SEQ IDNO: G002A, T006K, N008S, S009A, P012T, 534 I013V, S022T, S027K, F028P,S030K, G032A, N033P, K035A, Q036S, S042N, S044L, S050Y, L051V, K052Q,K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, E083L D_D0414327SEQ ID NO: G002A, T006K, N008S, S009A, P012T, 535 S022T, S027K, F028P,S030K, G032A, N033P, K035A, Q036S, S042N, S044L, L049M, S050Y, L051V,K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, E083L D_D0414355SEQ ID NO: G002A, T006K, N008S, S009A, P012T, 536 A016S, S022T, S027K,F028P, S030K, G032A, N033P, K035A, Q036S, S042N, S044L, L049M, S050Y,L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,E083L D_D0414366 SEQ ID NO: G002A, T006K, N008S, S009A, P012T, 537A016S, S022T, S027K, F028P, S030K, G032A, N033P, K035A, Q036S, E037D,T038S, S042N, S044L, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A,H058T, Q063L, A064S, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, E083L D_D0414388 SEQ ID NO: G002A, T004S,T006K, N008S, P012T, 538 S022T, S027K, F028P, S030K, G032A, N033P,K035A, Q036S, S042N, S044L, S050Y, L051V, K052Q, K053L, N054G, A056S,Q057A, H058T, Q063L, A064S, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, E083L D_D0414411 SEQ ID NO: G002A,T006K, P012T, A016S, S022T, 539 S027K, F028P, S030K, V031M, G032A,N033P, K035A, Q036S, T038S, S042N, S044L, L049M, S050Y, L051V, K052Q,K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L,E083L D_D0414412 SEQ ID NO: G002A, T006K, S009A, P012T, S022T, 540S027K, F028P, S030K, V031M, G032A, N033P, K035A, Q036S, T038S, S042N,S044L, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L,A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T,Q078S, R080Q, L081T, I082L, E083L D_D0414416 SEQ ID NO: G002A, T004S,T006K, S009A, P012T, 541 I013V, A016S, S022T, S027K, F028P, S030K,V031M, G032A, N033P, K035A, Q036S, E037D, S042N, S044L, L049M, S050Y,L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T,K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q,L081T, I082L, E083L D_D0414422 SEQ ID NO: G002A, T004S, T006K, N008S,S009A, 542 P012T, I013V, A016S, S022T, S027K, F028P, S030K, V031M,G032A, N033P, K035A, Q036S, T038S, S042N, S044L, L049M, S050Y, L051V,K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,I082L, E083L D_D0414424 SEQ ID NO: G002A, T004S, T006K, N008S, P012T,543 A016S, S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, E037D, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L, N054G,A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414425SEQ ID NO: G002A, T004S, T006K, N008S, S009A, 544 P012T, I013V, A016S,S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A, Q036S, E037D,S042N, S044L, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414427 SEQ ID NO: G002A,T006K, N008S, P012T, S022T, 545 S027K, F028P, S030K, V031M, G032A,N033P, K035A, Q036S, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L,N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0414430 SEQ ID NO: G002A, T004S, T006K, N008S, S009A, 546 P012T,I013V, A016S, S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, E037D, T038S, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L,N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0414433 SEQ ID NO: G002A, T004S, T006K, N008S, P012T, 547 A016S,S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A, Q036S, S042N,S044L, L049M, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414439 SEQ ID NO: G002A,T004S, T006K, N008S, S009A, 548 P012T, I013V, A016S, S022T, S027K,F028P, S030K, V031M, G032A, N033P, K035A, Q036S, S042N, S044L, S050Y,L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T,K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q,L081T, I082L, E083L D_D0414449 SEQ ID NO: G002A, T006K, N008S, S009A,P012T, 549 I013V, A016S, S022T, S027K, F028P, S030K, V031M, G032A,N033P, K035A, Q036S, T038S, S042N, S044L, L049M, S050Y, L051V, K052Q,K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L,E083L D_D0414450 SEQ ID NO: G002A, T004S, T006K, N008S, S009A, 550P012T, A016S, S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, T038S, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L, N054G,A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414453SEQ ID NO: G002A, T004S, T006K, N008S, S009A, 551 P012T, I013V, A016S,S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A, Q036S, S042N,S044L, L049M, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414454 SEQ ID NO: G002A,T004S, T006K, N008S, S009A, 552 P012T, I013V, A016S, S022T, S027K,F028P, S030K, V031M, G032A, N033P, K035A, Q036S, E037D, T038S, S042N,S044L, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L,A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T,Q078S, R080Q, L081T, I082L, E083L D_D0414455 SEQ ID NO: G002A, T006K,S009A, P012T, I013V, 553 S022T, S027K, F028P, S030K, V031M, G032A,N033P, K035A, Q036S, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L,N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0414459 SEQ ID NO: G002A, T004S, T006K, P012T, A016S, 554 S022T,S027K, F028P, S030K, V031M, G032A, N033P, K035A, Q036S, E037D, T038S,S042N, S044L, L049M, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A,H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414462 SEQ ID NO:G002A, T004S, T006K, N008S, S009A, 555 P012T, A016S, S022T, S027K,F028P, S030K, V031M, G032A, N033P, K035A, Q036S, E037D, T038S, S042N,S044L, L049M, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414466 SEQ ID NO: G002A,T004S, T006K, N008S, P012T, 556 I013V, S022T, S027K, F028P, S030K,V031M, G032A, N033P, K035A, Q036S, E037D, T038S, S042N, S044L, S050Y,L051V, K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T,K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q,L081T, I082L, E083L D_D0414470 SEQ ID NO: G002A, T006K, N008S, S009A,P012T, 557 S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, E037D, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L, N054G,A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414471SEQ ID NO: G002A, T004S, T006K, N008S, S009A, 558 P012T, I013V, A016S,S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A, Q036S, E037D,S042N, S044L, S050Y, L051V, K052Q, K053L, N054G, A056S, Q057A, H058T,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414475 SEQ ID NO: G002A,T004S, T006K, P012T, I013V, 559 S022T, S027K, F028P, S030K, V031M,G032A, N033P, K035A, Q036S, T038S, S042N, S044L, L049M, S050Y, L051V,K052Q, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,I082L, E083L D_D0414479 SEQ ID NO: G002A, T004S, T006K, N008S, P012T,560 I013V, S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, T038S, S042N, S044L, L049M, S050Y, L051V, K052Q, K053L, N054G,A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0414486SEQ ID NO: G002A, T006K, N008S, S009A, P012T, 561 I013V, S022T, S027K,F028P, S030K, V031M, G032A, N033P, K035A, Q036S, E037D, S042N, S044L,L049M, S050Y, L051V, K053L, N054G, A056S, Q057A, H058T, Q063L, A064S,S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S,R080Q, L081T, I082L, E083L D_D0414489 SEQ ID NO: G002A, T004S, T006K,N008S, S009A, 562 P012T, I013V, A016S, S022T, S027K, F028P, S030K,V031M, G032A, N033P, K035A, Q036S, E037D, S042N, S044L, S050Y, L051V,K053L, N054G, A056S, Q057A, H058T, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L,E083L D_D0414493 SEQ ID NO: G002A, T004S, T006K, N008S, P012T, 563I013V, A016S, S022T, S027K, F028P, S030K, V031M, G032A, N033P, K035A,Q036S, E037D, S042N, S044L, S050Y, L051V, K052Q, K053L, N054G, A056S,Q057A, H058T, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D,N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419050 SEQ IDNO: G002A, T004S, N008S, S009A, I013V, 564 S022T, G032A, T038S, S044L,L049M, L051V, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D,N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419051 SEQ IDNO: G002A, T004S, S009A, I013V, S022T, 565 G032A, E037D, T038S, S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, E083L D_D0419053 SEQ ID NO:G002A, T004S, S009A, S022T, G032A, 566 T038S, S044L, L049M, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419057 SEQ ID NO:G002A, T004AS, S009A, I013V, S022T, 567 G032A, E037D, T038S, S044L,L049M, L051V, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D,N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419058 SEQ IDNO: G002A, T004S, S009A, S022T, G032A, 568 S044L, L049M, L051V, K052Q,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419060 SEQ ID NO: G002A,T004S, N008S, S009A, I013V, 569 S022T, G032A, E037D, T038S, S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419062SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 570 G032A, S044L, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419064 SEQ ID NO:G002A, T004S, S009A, I013V, S022T, 571 G032A, T038S, S044L, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419066 SEQ ID NO:G002A, T004S, S009A, I013V, A016S, 572 S022T, G032A, E037D, T038S,S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0419071 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 573 G032A,E037D, T038S, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,I082L D_D0419072 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 574G032A, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L,E083L D_D0419075 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 575G032A, T038S, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,I082L, E083L D_D0419077 SEQ ID NO: G002A, T004S, S022T, G032A, E037D,576 T038S, S044L, L051V, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0419082 SEQ ID NO: G002A, T004S, S022T, G032A, E037D, 577 S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419083SEQ ID NO: G002A, T004S, S009A, S022T, G032A, 578 E037D, T038S, S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419084SEQ ID NO: G002A, T004S, N008S, S009A, I013V, 579 A016S, S022T, G032A,E037D, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L,E083L D_D0419087 SEQ ID NO: G002A, T004S, S009A, I013V, A016S, 580S022T, G032A, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,E083L D_D0419091 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 581G032A, T038S, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T,I082L, E083L D_D0419093 SEQ ID NO: G002A, I013V, S022T, G032A, S044L,582 L051V, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D,N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L, L085A D_D0419095SEQ ID NO: G002A, T004S, S009A, I013V, A016S, 583 S022T, G032A, S044L,L049M, L051V, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D,N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419096 SEQ IDNO: G002A, T004S, S009A, S022T, G032A, 584 T038S, S044L, L049M, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, E083L, L085A D_D0419098 SEQ ID NO: G002A,T004S, S009A, I013V, A016S, 585 S022T, G032A, E037D, T038S, S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q D_D0419099 SEQ ID NO: G002A,T004S, S022T, G032A, E037D, 586 T038S, S044L, L049M, L051V, K052Q,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419101 SEQ ID NO: G002A,T004S, S009A, S022T, G032A, 587 T038S, S044L, L049M, L051V, K052Q,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419102 SEQ ID NO: G002A,T004S, N008S, S009A, I013V, 588 S022T, G032A, S044L, L049M, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419106 SEQ ID NO:G002A, T004S, S009A, A016S, S022T, 589 G032A, S044L, L049M, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082L D_D0419108 SEQ ID NO: G002A,T004S, S009A, I013V, S022T, 590 G032A, E037D, T038S, S044L, L049M,L051V, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, E083L D_D0419109 SEQ ID NO: G002A,T004S, S009A, I013V, S022T, 591 G032A, E037D, S044L, L049M, L051V,K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D,A074K, K076T, Q078S, R080Q, L081T, I082F, E083V, L085V D_D0419110 SEQ IDNO: G002A, N008S, S009A, I013V, S022T, 592 G032A, E037D, T038S, S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419111SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 593 G032A, E037D, S044L,L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I, D071Y,N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419114SEQ ID NO: G002A, T004S, N008S, I013V, S022T, 594 G032A, E037D, T038S,S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0419115 SEQ ID NO: G002A, T004S, S009A, S022T, G032A, 595 E037D,T038S, S044L, L049M, L051V, Q063L, A064S, S065T, K067N, E069V, V070I,D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, I082L, E083LD_D0419117 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 596 G032A,T038S, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q D_D0419118 SEQ IDNO: G002A, T004S, S009A, I013V, S022T, 597 G032A, T038S, S044L, L051V,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q D_D0419119 SEQ ID NO: G002A, S009A, I013V, A016S,S022T, 598 G032A, T038S, S044L, L049M, L051V, K052Q, Q063L, A064S,S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S,R080Q, L081T, I082L, E083L, S086N D_D0419120 SEQ ID NO: G002A, N008S,S009A, I013V, S022T, 599 G032A, E037D, T038S, S044L, L049M, L051V,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L D_D0419124 SEQ ID NO: G002A,T004S, S009A, I013V, S022T, 600 G032A, S044L, L051V, Q063L, A064S,S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S,R080Q, L081T, I082L, E083L D_D0419126 SEQ ID NO: G002A, T004S, S022T,G032A, E037D, 601 T038S, S044L, L049M, L051V, K052Q, Q063L, A064S,S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S,R080Q, L081T D_D0419127 SEQ ID NO: G002A, T004S, S009A, I013V, S022T,602 G032A, E037D, S044L, L049M, L051V, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, I082L,E083L D_D0419128 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 603G032A, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T, K067N, E069V,V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, L081T, E083L,S086N D_D0419132 SEQ ID NO: G002A, T004S, S009A, I013V, S022T, 604G032A, E037D, T038S, S044L, L049M, L051V, Q063L, A064S, S065T, K067N,E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q, I082LD_D0419138 SEQ ID NO: G002A, T004S, S009A, I013V, A016S, 605 S022T,G032A, E037D, T038S, S044L, L049M, L051V, K052Q, Q063L, A064S, S065T,K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S, R080Q,I082L D_D0419142 SEQ ID NO: G002A, T004S, N008S, I013V, A016S, 606S022T, G032A, E037D, T038S, S044L, L049M, L051V, K052Q, Q063L, A064S,S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K, K076T, Q078S,R080Q, L081T, I082L, E083L, L085A D_D0419145 SEQ ID NO: G002A, T004S,S009A, I013V, S022T, 607 G032A, E037D, T038S, S044L, L051V, K052Q,Q063L, A064S, S065T, K067N, E069V, V070I, D071Y, N072D, N073D, A074K,K076T, Q078S, R080Q, L081T, I082L, E083L

Example 9—PIP-72Aa Variants with Multiple Amino Acid Substitutions

To generate further PIP-72 polypeptide variants site-directedmutagenesis was performed using the oligonucleotide primers listed inTable 19 to introduce amino acid substitutions from PIP-72 Da (SEQ IDNO: 10) into PIP-72Aa (SEQ ID NO: 2) or from PIP-72Aa (SEQ ID NO: 2)into PIP-72 Da (SEQ ID NO: 845—resynthesized to facilitate mutagenesis).An amino acid sequence alignment of PIP-72Aa (SEQ ID NO: 2) and PIP-72Da (SEQ ID NO: 10) is shown in FIG. 6. Synthetic gene fragments weresubcloned in expression vector pCOLD™1 as described in Example 3.

TABLE 19 Sequence Sequence Primer identifier Primer identifier 72_DG_1aFSEQ ID NO: 171 72_DG_5aF-2 SEQ ID NO: 181 72_DG_1bF SEQ ID NO: 17272_DG_5bF-2 SEQ ID NO: 182 72_DG_1cF SEQ ID NO: 173 72_DG_5cF-2 SEQ IDNO: 183 72_DG_1dF SEQ ID NO: 174 72_DG_5dF-2 SEQ ID NO: 184 72_DG_2F SEQID NO: 175 72c_Div_1F SEQ ID NO: 185 72_DG_3aF SEQ ID NO: 176 72c_Div_2FSEQ ID NO: 186 72_DG_3bF SEQ ID NO: 177 72c_Div_3F SEQ ID NO: 18772_DG_3cF SEQ ID NO: 178 72c_Div_4F SEQ ID NO: 188 72_DG_3dF SEQ ID NO:179 72c_Div_5F-2 SEQ ID NO: 189 72_DG_4F SEQ ID NO: 180

The resulting plasmid DNAs were transformed into E. coli, and theinsecticidal protein was expressed as described in Example 3. Bioassayswere performed on Western Corn Root Worm as described in Example 4 todetermine which gene variants retained insecticidal activity. DNAsequencing was performed on the active gene variants. The percentidentity compared to PIP-72Aa (SEQ ID NO: 2), clone designation,nucleotide and amino acid sequences of the resulting active PIP-72polypeptide variants are listed in Table 20. Out of approximately 180unique PIP-72 polypeptide variants that were screened, 156 activevariants were identified. Table 21 summarizes the % identity of theactive variants compared to PIP-72Aa (SEQ ID NO: 17), the number ofclones with each % identity and the clone identification. Table 22 showsthe amino acid substitutions of the resulting active PIP-72 polypeptidevariants compared to PIP-72Aa (SEQ ID NO: 2).

TABLE 20 % Identity to PIP-72Aa Clone SEQ ID NO: 2 designationsPolynucleotide Polypeptide 86% D_D0403719 SEQ ID NO: 367 SEQ ID NO: 60886% D_D0403734 SEQ ID NO: 368 SEQ ID NO: 609 86% D_D0403823 SEQ ID NO:369 SEQ ID NO: 610 86% D_D0403844 SEQ ID NO: 370 SEQ ID NO: 611 86%D_D0403865 SEQ ID NO: 371 SEQ ID NO: 612 86% D_D0403897 SEQ ID NO: 372SEQ ID NO: 613 86% D_D0403907 SEQ ID NO: 373 SEQ ID NO: 614 95%D_D0404048 SEQ ID NO: 374 SEQ ID NO: 615 98% D_D0404051 SEQ ID NO: 375SEQ ID NO: 616 96% D_D0404052 SEQ ID NO: 376 SEQ ID NO: 617 98%D_D0404054 SEQ ID NO: 377 SEQ ID NO: 618 95% D_D0404056 SEQ ID NO: 378SEQ ID NO: 619 98% D_D0404057 SEQ ID NO: 379 SEQ ID NO: 620 96%D_D0404062 SEQ ID NO: 380 SEQ ID NO: 621 95% D_D0404066 SEQ ID NO: 381SEQ ID NO: 622 96% D_D0404067 SEQ ID NO: 382 SEQ ID NO: 623 95%D_D0404068 SEQ ID NO: 383 SEQ ID NO: 624 95% D_D0404073 SEQ ID NO: 384SEQ ID NO: 625 98% D_D0404074 SEQ ID NO: 385 SEQ ID NO: 626 95%D_D0404078 SEQ ID NO: 386 SEQ ID NO: 627 88% D_D0404079 SEQ ID NO: 387SEQ ID NO: 628 98% D_D0404098 SEQ ID NO: 388 SEQ ID NO: 629 92%D_D0404103 SEQ ID NO: 389 SEQ ID NO: 630 98% D_D0404111 SEQ ID NO: 390SEQ ID NO: 631 99% D_D0404113 SEQ ID NO: 391 SEQ ID NO: 632 99%D_D0404116 SEQ ID NO: 392 SEQ ID NO: 633 98% D_D0404119 SEQ ID NO: 393SEQ ID NO: 634 96% D_D0404120 SEQ ID NO: 394 SEQ ID NO: 635 96%D_D0404121 SEQ ID NO: 395 SEQ ID NO: 636 96% D_D0404122 SEQ ID NO: 396SEQ ID NO: 637 96% D_D0404128 SEQ ID NO: 397 SEQ ID NO: 638 98%D_D0404130 SEQ ID NO: 398 SEQ ID NO: 639 95% D_D0404135 SEQ ID NO: 399SEQ ID NO: 640 91% D_D0405129 SEQ ID NO: 400 SEQ ID NO: 641 92%D_D0405130 SEQ ID NO: 401 SEQ ID NO: 642 94% D_D0405132 SEQ ID NO: 402SEQ ID NO: 643 95% D_D0405135 SEQ ID NO: 403 SEQ ID NO: 644 90%D_D0405137 SEQ ID NO: 404 SEQ ID NO: 645 94% D_D0405139 SEQ ID NO: 405SEQ ID NO: 646 87% D_D0405141 SEQ ID NO: 406 SEQ ID NO: 647 85%D_D0405142 SEQ ID NO: 407 SEQ ID NO: 648 92% D_D0405145 SEQ ID NO: 408SEQ ID NO: 649 99% D_D0405148 SEQ ID NO: 409 SEQ ID NO: 650 98%D_D0405151 SEQ ID NO: 410 SEQ ID NO: 651 98% D_D0405153 SEQ ID NO: 411SEQ ID NO: 652 98% D_D0405157 SEQ ID NO: 412 SEQ ID NO: 653 96%D_D0405159 SEQ ID NO: 413 SEQ ID NO: 654 99% D_D0405163 SEQ ID NO: 414SEQ ID NO: 655 87% D_D0405169 SEQ ID NO: 415 SEQ ID NO: 656 99%D_D0405174 SEQ ID NO: 416 SEQ ID NO: 657 90% D_D0405177 SEQ ID NO: 417SEQ ID NO: 658 93% D_D0405183 SEQ ID NO: 418 SEQ ID NO: 659 88%D_D0405187 SEQ ID NO: 419 SEQ ID NO: 660 95% D_D0405191 SEQ ID NO: 420SEQ ID NO: 661 93% D_D0405192 SEQ ID NO: 421 SEQ ID NO: 662 93%D_D0405193 SEQ ID NO: 422 SEQ ID NO: 663 94% D_D0405199 SEQ ID NO: 423SEQ ID NO: 664 91% D_D0405204 SEQ ID NO: 424 SEQ ID NO: 665 93%D_D0405205 SEQ ID NO: 425 SEQ ID NO: 666 98% D_D0405213 SEQ ID NO: 426SEQ ID NO: 667 82% D_D0405218 SEQ ID NO: 427 SEQ ID NO: 668 82%D_D0405219 SEQ ID NO: 428 SEQ ID NO: 669 98% D_D0405227 SEQ ID NO: 429SEQ ID NO: 670 96% D_D0405228 SEQ ID NO: 430 SEQ ID NO: 671 99%D_D0405230 SEQ ID NO: 431 SEQ ID NO: 672 96% D_D0405231 SEQ ID NO: 432SEQ ID NO: 673 99% D_D0405233 SEQ ID NO: 433 SEQ ID NO: 674 94%D_D0405238 SEQ ID NO: 434 SEQ ID NO: 675 94% D_D0405239 SEQ ID NO: 435SEQ ID NO: 676 96% D_D0405241 SEQ ID NO: 436 SEQ ID NO: 677 96%D_D0405242 SEQ ID NO: 437 SEQ ID NO: 678 98% D_D0405243 SEQ ID NO: 438SEQ ID NO: 679 96% D_D0405244 SEQ ID NO: 439 SEQ ID NO: 680 99%D_D0405249 SEQ ID NO: 440 SEQ ID NO: 681 96% D_D0405251 SEQ ID NO: 441SEQ ID NO: 682 98% D_D0405252 SEQ ID NO: 442 SEQ ID NO: 683 99%D_D0405254 SEQ ID NO: 443 SEQ ID NO: 684 99% D_D0405258 SEQ ID NO: 444SEQ ID NO: 685 98% D_D0405260 SEQ ID NO: 445 SEQ ID NO: 686 98%D_D0405261 SEQ ID NO: 446 SEQ ID NO: 687 98% D_D0405281 SEQ ID NO: 447SEQ ID NO: 688 93% D_D0405282 SEQ ID NO: 448 SEQ ID NO: 689 94%D_D0405284 SEQ ID NO: 449 SEQ ID NO: 690 96% D_D0405285 SEQ ID NO: 450SEQ ID NO: 691 95% D_D0405288 SEQ ID NO: 451 SEQ ID NO: 692 95%D_D0405289 SEQ ID NO: 452 SEQ ID NO: 693 95% D_D0405290 SEQ ID NO: 453SEQ ID NO: 694 98% D_D0405291 SEQ ID NO: 454 SEQ ID NO: 695 95%D_D0405292 SEQ ID NO: 455 SEQ ID NO: 696 99% D_D0405294 SEQ ID NO: 456SEQ ID NO: 697 94% D_D0405297 SEQ ID NO: 457 SEQ ID NO: 698 95%D_D0405299 SEQ ID NO: 458 SEQ ID NO: 699 94% D_D0405300 SEQ ID NO: 459SEQ ID NO: 700 99% D_D0405302 SEQ ID NO: 460 SEQ ID NO: 701 94%D_D0405307 SEQ ID NO: 461 SEQ ID NO: 702 91% D_D0405309 SEQ ID NO: 462SEQ ID NO: 703 99% D_D0405310 SEQ ID NO: 463 SEQ ID NO: 704 94%D_D0405311 SEQ ID NO: 464 SEQ ID NO: 705 96% D_D0405312 SEQ ID NO: 465SEQ ID NO: 706 94% D_D0405313 SEQ ID NO: 466 SEQ ID NO: 707 96%D_D0405320 SEQ ID NO: 467 SEQ ID NO: 708 70% D_D0408199 SEQ ID NO: 468SEQ ID NO: 709 70% D_D0408201 SEQ ID NO: 469 SEQ ID NO: 710 71%D_D0408212 SEQ ID NO: 470 SEQ ID NO: 711 76% D_D0408220 SEQ ID NO: 471SEQ ID NO: 712 77% D_D0408221 SEQ ID NO: 472 SEQ ID NO: 713 73%D_D0408223 SEQ ID NO: 473 SEQ ID NO: 714 77% D_D0408226 SEQ ID NO: 474SEQ ID NO: 715 73% D_D0408229 SEQ ID NO: 475 SEQ ID NO: 716 72%D_D0408231 SEQ ID NO: 476 SEQ ID NO: 717 73% D_D0408234 SEQ ID NO: 477SEQ ID NO: 718 72% D_D0408238 SEQ ID NO: 478 SEQ ID NO: 719 72%D_D0408241 SEQ ID NO: 479 SEQ ID NO: 720 71% D_D0408246 SEQ ID NO: 480SEQ ID NO: 721 76% D_D0408250 SEQ ID NO: 481 SEQ ID NO: 722 73%D_D0408253 SEQ ID NO: 482 SEQ ID NO: 723 72% D_D0408254 SEQ ID NO: 483SEQ ID NO: 724 71% D_D0408261 SEQ ID NO: 484 SEQ ID NO: 725 70%D_D0408267 SEQ ID NO: 485 SEQ ID NO: 726 72% D_D0408268 SEQ ID NO: 486SEQ ID NO: 727 73% D_D0408270 SEQ ID NO: 487 SEQ ID NO: 728 70%D_D0408275 SEQ ID NO: 488 SEQ ID NO: 729 72% D_D0408276 SEQ ID NO: 489SEQ ID NO: 730 74% D_D0408277 SEQ ID NO: 490 SEQ ID NO: 731 78%D_D0408283 SEQ ID NO: 491 SEQ ID NO: 732 74% D_D0408284 SEQ ID NO: 492SEQ ID NO: 733 73% D_D0408286 SEQ ID NO: 493 SEQ ID NO: 734 76%D_D0408287 SEQ ID NO: 494 SEQ ID NO: 735 77% D_D0408294 SEQ ID NO: 495SEQ ID NO: 736 73% D_D0408295 SEQ ID NO: 496 SEQ ID NO: 737 79%D_D0408296 SEQ ID NO: 497 SEQ ID NO: 738 74% D_D0408297 SEQ ID NO: 498SEQ ID NO: 739 71% D_D0408298 SEQ ID NO: 499 SEQ ID NO: 740 74%D_D0408302 SEQ ID NO: 500 SEQ ID NO: 741 74% D_D0408308 SEQ ID NO: 501SEQ ID NO: 742 73% D_D0408309 SEQ ID NO: 502 SEQ ID NO: 743 72%D_D0408314 SEQ ID NO: 503 SEQ ID NO: 744 74% D_D0408318 SEQ ID NO: 504SEQ ID NO: 745 73% D_D0408319 SEQ ID NO: 505 SEQ ID NO: 746 72%D_D0408322 SEQ ID NO: 506 SEQ ID NO: 747 79% D_D0408329 SEQ ID NO: 507SEQ ID NO: 748 78% D_D0408331 SEQ ID NO: 508 SEQ ID NO: 749 74%D_D0408338 SEQ ID NO: 509 SEQ ID NO: 750 78% D_D0408340 SEQ ID NO: 510SEQ ID NO: 751 70% D_D0408353 SEQ ID NO: 511 SEQ ID NO: 752 78%D_D0408357 SEQ ID NO: 512 SEQ ID NO: 753 76% D_D0408359 SEQ ID NO: 513SEQ ID NO: 754 73% D_D0408361 SEQ ID NO: 514 SEQ ID NO: 755 78%D_D0408366 SEQ ID NO: 515 SEQ ID NO: 756 74% D_D0408368 SEQ ID NO: 516SEQ ID NO: 757 73% D_D0408370 SEQ ID NO: 517 SEQ ID NO: 758 73%D_D0408372 SEQ ID NO: 518 SEQ ID NO: 759 74% D_D0408373 SEQ ID NO: 519SEQ ID NO: 760 72% D_D0408374 SEQ ID NO: 520 SEQ ID NO: 761 73%D_D0408377 SEQ ID NO: 521 SEQ ID NO: 762 72% D_D0408381 SEQ ID NO: 522SEQ ID NO: 763 74% D_D0408382 SEQ ID NO: 523 SEQ ID NO: 764 73%D_D0408384 SEQ ID NO: 524 SEQ ID NO: 765 76% D_D0408385 SEQ ID NO: 525SEQ ID NO: 766 72% D_D0408386 SEQ ID NO: 526 SEQ ID NO: 767 74%D_D0408387 SEQ ID NO: 527 SEQ ID NO: 768 78% D_D0348839 SEQ ID NO: 770SEQ ID NO: 772 85% D_D0347298 SEQ ID NO: 769 SEQ ID NO: 771

TABLE 21 Iden- tity to PIP- 72Aa # of (%) variants Variants name 46% 1D_D0414430 48% 4 D_D0407437, D_D0414422, D_D0414454, D_D0414462 49% 6D_D0414416, D_D0414425, D_D0414449, D_D0414450, D_D0414453, D_D041447150% 7 D_D0414424, D_D0414439, D_D0414459, D_D0414466, D_D0414479,D_D0414489, D_D0414493 51% 4 D_D0414433, D_D0414470, D_D0414475,D_D0414486 52% 2 D_D0414411, D_D0414455 53% 4 D_D0407429, D_D0414366,D_D0414412, D_D0414427 55% 2 D_D0414317, D_D0414355 56% 2 D_D0407455,D_D0414326 57% 3 D_D0407456, D_D0414327, D_D0414388 65% 1 D_D0419142 66%3 D_D0419060, D_D0419066, D_D0419084 67% 5 D_D0407461, D_D0419109,D_D0419110, D_D0419114, D_D0419119 69% 11 D_D0419050, D_D0419057,D_D0419071, D_D0419075, D_d0419083, D_D0419091, D_D0419102, D_D0419111,D_D0419120, D_D0419138, D_D0419145 70% 17 D_D0419051, D_D0419053,D_D0419064, D_D0419072, D_D0419087, D_D0419095, D_D0419098, D_D0419099,D_D0419101, D_D0419108, D_D0419115, D_D0419128, D_D0408199, D_D0408201,D_D0408267, D_D0408275, D_D0408353 71% 11 D_D0419058, D_D0419062,D_D0419082, D_D0419096, D_D0419106, D_D0419127, D_D0419132, D_D0408212,D_D0408246, D_D0408261, D_D0408298 72% 15 D_D0419077, D_D0419117,D_D0419124, D_D0419126, D_D0408231, D_D0408238, D_D0408241, D_D0408254,D_D0408268, D_D0408276, D_D0408314, D_D0408322, D_D0408374, D_D0408381,D_D0408386 73% 15 D_D0419093, D_D0408223, D_D0408229, D_D0408234,D_D0408253, D_D0408270, D_D0408286, D_D0408295, D_D0408309, D_D0408319,D_D0408361, D_D0408370, D_D0408372, D_D0408377, D_D0408384 74% 12D_D0419118, D_D0408277, D_D0408284, D_D0408297, D_D0408302, D_D0408308,D_D0408318, D_D0408338, D_D0408368, D_D0408373, D_D0408382, D_D040838776% 5 D_D0408220, D_D0408250, D_D0408287, D_D0408359, D_D0408385 77% 3D_D0408221, D_D0408226, D_D0408294 78% 6 D_D0408283, D_D0408331,D_D0408340, D_D0408357, D_D0408366, D_D0348839 79% 2 D_D0408296,D_D0408329 82% 2 D_D0405218, D_D0405219 85% 2 D_D0405142, D_D0347298 86%7 D_D0403719, D_D0403734, D_D0403823, D_D0403844, D_D0403865,D_D0403897, D_D0403907 87% 2 D_D0405141, D_D0405169 88% 2 D_D0404079,D_D0405187 90% 2 D_D0405137, D_D0405177 91% 3 D_D0405129, D_D0405204,D_D0405309 92% 3 D_D0404103, D_D0405130, D_D0405145 93% 5 D_D0405183,D_D0405192, D_D0405193, D_D0405205, D_D0405282 94% 11 D_D0405132,D_D0405139, D_D0405199, D_D0405238, D_D0405239, D_D0405284, D_D0405297,D_D0405300, D_D0405307, D_D0405311, D_D0405313 95% 14 D_D0404048,D_D0404056, D_D0404066, D_D0404068, D_D0404073, D_D0404078, D_D0404135,D_D0405135, D_D0405191, D_D0405288, D_D0405289, D_D0405290, D_D0405292,D_D0405299 96% 17 D_D0404052, D_D0404062, D_D0404067, D_D0404120,D_D0404121, D_D0404122, D_D0404128, D_D0405159, D_D0405228, D_D0405231,D_D0405241, D_D0405242, D_D0405244, D_D0405251, D_D0405285, D_D0405312,D_D0405320 98% 19 D_D0404051, D_D0404054, D_D0404057, D_D0404074,D_D0404098, D_D0404111, D_D0404119, D_D0404130, D_D0405151, D_D0405153,D_D0405157, D_D0405213, D_D0405227, D_D0405243, D_D0405252, D_D0405260,D_D0405261, D_D0405281, D_D0405291 99% 13 D_D0404113, D_D0404116,D_D0405148, D_D0405163, D_D0405174, D_D0405230, D_D0405233, D_D0405249,D_D0405254, D_D0405258, D_D0405294, D_D0405302, D_D0405310

TABLE 22 Clone Poly- Amino Acid Substitutions compared Designationpeptide to PIP-72Aa (SEQ ID NO: 2) D_D0403719 SEQ ID N011G, S027Q,S030P, N033S, T038S, S044D, NO: 608 K053N, D071E, A074Y, Q078S, R080E,E083L D_D0403734 SEQ ID N011G, P012D, S027Q, S030K, G032A, N033P, NO:609 T038S, S044A, K053N, D071E, R080Q, E083L D_D0403823 SEQ ID N011G,P012D, S030K, G032A, N033P, S044D, NO: 610 K053N, D071E, N073D, A074Y,R080Q, E083L D_D0403844 SEQ ID S009L, P012D, S027Q, S030K, N033P, T038S,NO: 611 S044A, D071E, N073D, Q078S, R080E, E083L D_D0403865 SEQ IDN008S, N011G, P012D, S030K, N033S, T038S, NO: 612 S044D, K053N, D071E,A074Y, Q078S, R080E D_D0403897 SEQ ID N011G, S027Q, S030K, G032A, N033P,T038S, NO: 613 S044D, K053N, D071E, N073D, Q078S, R080E D_D0403907 SEQID N008S, N011G, P012D, S027Q, S030K, N033S, NO: 614 S044D, K053N,D071E, N073D, Q078S, R080Q D_D0404048 SEQ ID N011K, V015A, K053R, H058ANO: 615 D_D0404051 SEQ ID T038S, K053R NO: 616 D_D0404052 SEQ ID N011K,R080Q, E083Q NO: 617 D_D0404054 SEQ ID N011K, T038S NO: 618 D_D0404056SEQ ID V015A, H019K, K053R, H058A NO: 619 D_D0404057 SEQ ID N011K, A074TNO: 620 D_D0404062 SEQ ID K053R, E083H, L085V NO: 621 D_D0404066 SEQ IDK053R, H058A, E083H, L085V NO: 622 D_D0404067 SEQ ID N011K, V015A, K053RNO: 623 D_D0404068 SEQ ID N033S, K053R, A074T, R080E NO: 624 D_D0404073SEQ ID T038S, K053R, H058A, L085V NO: 625 D_D0404074 SEQ ID K053R, E083HNO: 626 D_D0404078 SEQ ID N011K, P012K, H019K, H058A NO: 627 D_D0404079SEQ ID N011K, S030G, G032D, T038S, K053R, S065T, NO: 628 D071E, A074T,L085V, S086A D_D0404098 SEQ ID S065T, V070I NO: 629 D_D0404103 SEQ IDV015A, H019K, V031I, G032D, N033S, T038S, NO: 630 K053R D_D0404111 SEQID T038S, K053R NO: 631 D_D0404113 SEQ ID K053N NO: 632 D_D0404116 SEQID E083H NO: 633 D_D0404119 SEQ ID P012K, H019K NO: 634 D_D0404120 SEQID S030G, N033S, T038S NO: 635 D_D0404121 SEQ ID H058A, R080E, L085V NO:636 D_D0404122 SEQ ID E083H, L085V, S086A NO: 637 D_D0404128 SEQ IDN011K, V015A, E083H NO: 638 D_D0404130 SEQ ID N011K, H019K NO: 639D_D0404135 SEQ ID S030G, V031I, N033S, T038S NO: 640 D_D0405129 SEQ IDN011K, P012K, V015A, H019K, S065T, V070I, NO: 641 D071E, A074TD_D0405130 SEQ ID H019K, K053R, H058A, S065T, V070I, D071E, NO: 642A074T D_D0405132 SEQ ID S030G, V031I, G032D, N033S, T038S NO: 643D_D0405135 SEQ ID N011K, P012K, V015A, H019K NO: 644 D_D0405137 SEQ IDN011K, P012K, V015A, H019K, S030G, V031I, NO: 645 G032D, N033S, T038SD_D0405139 SEQ ID N011K, P012K, V015A, H019K, K053S NO: 646 D_D0405141SEQ ID N011K, P012K, V015A, H019K, T038S, K053R, NO: 647 H058A, S065T,V070I, D071E, A074T D_D0405142 SEQ ID N011K, P012K, V015A, H019K, S030G,V031I, NO: 648 G032D, N033S, T038S, S065T, V070I, D071E, A074TD_D0405145 SEQ ID S030G, V031I, G032D, N033S, T038S, K053R, NO: 649H058A D_D0405148 SEQ ID T038S NO: 650 D_D0405151 SEQ ID V015A, H019K NO:651 D_D0405153 SEQ ID K053R, H058A NO: 652 D_D0405157 SEQ ID T038S,H058A NO: 653 D_D0405159 SEQ ID K053R, H058A, A074T NO: 654 D_D0405163SEQ ID K053R NO: 655 D_D0405169 SEQ ID N011K, P012K, V015A, H019K,S030G, V031I, NO: 656 G032D, N033S, T038S, K053R, H058A D_D0405174 SEQID H058A NO: 657 D_D0405177 SEQ ID S030G, V031I, G032D, N033S, T038S,S065T, NO: 658 V070I, D071E, A074T D_D0405183 SEQ ID N011K, P012K,V015A, H019K, T0385, H058A NO: 659 D_D0405187 SEQ ID N011K, P012K,V015A, H019K, S030G, V031I, NO: 660 G032D, N033S, K053R, H058AD_D0405191 SEQ ID S065T, V070I, D071E, A074T NO: 661 D_D0405192 SEQ IDK053R, H058A, S065T, V070I, D071E, A074T NO: 662 D_D0405193 SEQ IDN011K, P012K, V015A, H019K, N033S, T038S NO: 663 D_D0405199 SEQ IDK053R, H058A, V070I, D071E, A074T NO: 664 D_D0405204 SEQ ID S030G,V031I, G032D, N033S, T038S, K053R, NO: 665 H058A, A074T D_D0405205 SEQID N011K, P012K, V015A, H019K, K053R, H058A NO: 666 D_D0405213 SEQ IDK053S, H058A NO: 667 D_D0405218 SEQ ID N011K, P012K, V015A, H019K,S030G, V031I, NO: 668 G032D, N033S, T038S, K053R, H058A, R080E, E083H,L085V, S086A D_D0405219 SEQ ID N011K, P012K, V015A, H019K, S030G, V031I,NO: 669 G032D, N033S, T038S, K053R, H058A, S065T, V070I, D071E, A074TD_D0405227 SEQ ID R080E, S086A NO: 670 D_D0405228 SEQ ID N011K, V015A,L085V NO: 671 D_D0405230 SEQ ID H019K NO: 672 D_D0405231 SEQ ID N011K,E083H, L085V NO: 673 D_D0405233 SEQ ID N011K NO: 674 D_D0405238 SEQ IDN011K, V015A, E083H, L085V, S086A NO: 675 D_D0405239 SEQ ID N011K,H058A, R080E, L085V, S086A NO: 676 D_D0405241 SEQ ID N011K, P012K, V015ANO: 677 D_D0405242 SEQ ID N011K, R080E, S086A NO: 678 D_D0405243 SEQ IDN011K, V015A NO: 679 D_D0405244 SEQ ID N011K, V015A, H019K NO: 680D_D0405249 SEQ ID P012K NO: 681 D_D0405251 SEQ ID P012K, V015A, H019KNO: 682 D_D0405252 SEQ ID P012K, V015A NO: 683 D_D0405254 SEQ ID V015ANO: 684 D_D0405258 SEQ ID R080E NO: 685 D_D0405260 SEQ ID H019K, K053RNO: 686 D_D0405261 SEQ ID E083H, L085V NO: 687 D_D0405281 SEQ ID N033S,T038S NO: 688 D_D0405282 SEQ ID S030G, V031I, G032D, N033S, T038S, K053RNO: 689 D_D0405284 SEQ ID V031I, G032D, T038S, R080E, L085V NO: 690D_D0405285 SEQ ID K053R, R080E, S086A NO: 691 D_D0405288 SEQ ID S030G,N033S, R080E, S086A NO: 692 D_D0405289 SEQ ID G032D, K053R, R080E, E083HNO: 693 D_D0405290 SEQ ID S030G, V031I, G032D, N033S NO: 694 D_D0405291SEQ ID S030G, K053R NO: 695 D_D0405292 SEQ ID N033S, K053R, R080E, S086ANO: 696 D_D0405294 SEQ ID S030G NO: 697 D_D0405297 SEQ ID S030G, V031I,G032D, N033S, K053R NO: 698 D_D0405299 SEQ ID K053H, H058P, E083H, S086ANO: 699 D_D0405300 SEQ ID G032D, N033S, T038S, L085V, S086A NO: 700D_D0405302 SEQ ID N033S NO: 701 D_D0405307 SEQ ID N033S, T038S, K053R,H058A, R080E NO: 702 D_D0405309 SEQ ID S030G, V031I, N033S, T038S,K053R, H058A, NO: 703 R080E, L085V D_D0405310 SEQ ID G032D NO: 704D_D0405311 SEQ ID S030G, G032D, N033S, L085V, S086A NO: 705 D_D0405312SEQ ID S030G, V031I, T038S NO: 706 D_D0405313 SEQ ID S030G, G032D,N033S, T038S, H058A NO: 707 D_D0405320 SEQ ID S030G, N033S, H058A NO:708 D_D0408199 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 709S027K, S030G, V031I, G032D, N033S, T038S, S044D, K053R, H058A, S065T,V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086AD_D0408201 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 710S027K, S030G, V031I, G032D, N033S, T038S, S044D, A056T, H058A, S065T,V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086AD_D0408212 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 711S027K, S030G, V031I, G032D, N033S, T038S, S044D, H058A, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408220SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 712 S027K, S030G,V031I, G032D, N033S, T038S, S044D, H058A, S065T, V070I, D071E, N073S,A074T, E083H, L085V D_D0408221 SEQ ID N011K, V015A, S027K, S030G, V031I,G032D, NO: 713 N033S, T038S, S044D, S065T, V070I, D071E, N073S, A074T,Q078H, R080E, L081T, E083H, L085V, S086A D_D0408223 SEQ ID N011K, V015A,H019K, S027K, S030G, V031I, NO: 714 G032D, N033S, T038S, S044D, K053R,H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H,L085V, S086A D_D0408226 SEQ ID N011K, V015A, H019K, S027K, S030G, V031I,NO: 715 G032D, N033S, T038S, S044D, K053R, H058A, S065T, V070I, D071E,N073S, A074T, R080E, E083H, L085V D_D0408229 SEQ ID N008K, N011K, P012K,V015A, A016S, H019K, NO: 716 S027K, S030G, V031I, G032D, N033S, T038S,S044D, K053R, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L085V,S086A D_D0408231 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO:717 S027K, S030G, V031I, G032D, N033S, T038S, S044D, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408234SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 718 S027K, S030G,V031I, G032D, N033S, T038S, S044D, K053R, A056T, H058A, S065T, V070I,D071E, N073S, A074T, R080E, S086A D_D0408238 SEQ ID P012K, V015A, H019K,S027K, S030G, V031I, NO: 719 G032D, N033S, T038S, S044D, K053R, A056T,H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H,L085V, S086A D_D0408241 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K,NO: 720 S027K, S030G, V031I, G032D, N033S, T038S, S044D, K053R, H058A,S065T, V070I, D071E, N073S, A074T, R080E, E083H, L085V, S086A D_D0408246SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 721 S022R, S027K,S030G, V031I, G032D, N033S, T038S, S044D, K053R, A056T, H058A, S065T,V070I, D071E, N073S, A074T, E083H, L085V, S086A D_D0408250 SEQ ID N008K,N011K, P012K, V015A, A016S, H019K, NO: 722 V031I, N033S, S044D, K053R,S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V,S086A D_D0408253 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO:723 S030G, N033S, S044D, K053R, A056T, H058A, S065T, V070I, D071E,N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408254 SEQ IDN008K, N011K, P012K, V015A, A016S, H019K, NO: 724 S030G, G032D, N033S,S044D, K053R, A056T, H058A, S065T, V070I, D071E, N073S, A074T, Q078H,R080E, L081T, E083H, L085V, S086A D_D0408261 SEQ ID N008K, N011K, P012K,V015A, A016S, H019K, NO: 725 S030G, G032D, N033S, T038S, S044D, K053R,A056T, H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T,E083H, L085V, S086A D_D0408267 SEQ ID N008K, N011K, P012K, V015A, A016S,H019K, NO: 726 S027K, S030G, V031I, G032D, N033S, T038S, K053R, A056T,H058A, S065T, V070I, D071E, N0735, A074T, Q078H, R080E, L081T, E083H,L085V, S086A D_D0408268 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K,NO: 727 S030G, N033S, T038S, S044D, K053R, A056T, H058A, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408270SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 728 V031I, G032D,N033S, S044D, K053R, S065T, S066N, V070I, D071E, N073S, A074T, Q078H,R080E, L081T, E083H, L085V, S086A D_D0408275 SEQ ID N008K, N011K, P012K,V015A, A016S, H019K, NO: 729 S027K, S030G, V031I, G032D, N033S, S044D,K053R, A056T, H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408276 SEQ ID N008K, N011K, P012K, V015A,A016S, H019K, NO: 730 S027K, S030G, V031I, G032D, N033S, K053R, H058A,S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V,S086A D_D0408277 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO:731 S027K, S030G, V031I, G032D, N033S, S044D, K053R, H058A, S065T,V070I, D071E, N073S, A074T, R080E, E083H, L085V D_D0408283 SEQ ID N008K,N011K, P012K, V015A, A016S, H019K, NO: 732 S027K, S030G, N033S, S044D,K053R, A056T, H058A, S065T, V070I, D071E, N073S, A074T, R080E D_D0408284SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 733 S030G, G032D,N033S, S044D, K053R, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408286 SEQ ID N008K, N011K, P012K, V015A,A016S, H019K, NO: 734 S027K, S030G, V031I, G032D, N033S, T038S, S044D,K053R, S065T, V070I, D071E, N073S, A074T, R080E, E083H, L085V, S086AD_D0408287 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 735N033S, S044D, K053R, H058A, S065T, V070I, D071E, N073S, A074T, Q078H,R080E, L081T, E083H, L085V, S086A D_D0408294 SEQ ID H019K, S027K, S030G,V031I, G032D, N033S, NO: 736 T038S, S044D, K053R, S065T, V070I, D071E,N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408295 SEQ IDP012K, V015A, H019K, S027K, S030G, V031I, NO: 737 G032D, N033S, T038S,S044D, K053R, H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408296 SEQ ID H019K, S027K, S030G, V031I,G032D, N033S, NO: 738 T038S, S044D, K053R, A056T, H058A, V070I, D071E,R080E, L081T, E083H, L085V, S086A D_D0408297 SEQ ID N008K, N011K, P012K,V015A, A016S, H019K, NO: 739 S027K, S030G, V031I, G032D, N033S, T038S,S044D, H058A, S065T, V070I, D071E, N073S, A074T, R080E, E083H, S086AD_D0408298 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 740S027K, S030G, V031I, G032D, N033S, T038S, S044D, K053R, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408302SEQ ID N011K, P012K, V015A, H019K, S027K, S030G, NO: 741 V031I, G032D,N033S, T038S, S044D, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408308 SEQ ID N011K, V015A, S027K, S030G,V031I, G032D, NO: 742 N033S, T038S, S044D, K053R, H058A, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408309SEQ ID N011K, V015A, S027K, S030G, V031I, G032D, NO: 743 N033S, T038S,S044D, K053R, A056T, H058A, S065T, V070I, D071E, N073S, A074T, Q078H,R080E, L081T, E083H, L085V, S086A D_D0408314 SEQ ID N008K, N011K, P012K,V015A, A016S, H019K, NO: 744 S030G, V031I, G032D, N033S, T038S, S044D,H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H,L085V, S086A D_D0408318 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K,NO: 745 S027K, S030G, V031I, G032D, N033S, T038S, S044D, K053R, S065T,D071E, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408319 SEQ ID N008K,N011K, P012K, V015A, A016S, H019K, NO: 746 S027K, S030G, V031I, G032D,N033S, T038S, S044D, K053R, S065T, V070I, A074T, Q078H, R080E, L081T,E083H, L085V, S086A D_D0408322 SEQ ID N011K, P012K, V015A, S027K, S030G,V031I, NO: 747 G032D, N033S, T038S, S044D, K053R, A056T, H058A, S065T,V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086AD_D0408329 SEQ ID N011K, V015A, H019K, S027K, S030G, V031I, NO: 748G032D, N033S, T038S, S044D, K053R, H058A, S065T, V070I, D071E, N073S,A074T, E083H D_D0408331 SEQ ID N011K, V015A, H019K, G032D, N033S, S044D,NO: 749 K053R, H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408338 SEQ ID N008K, N011K, P012K, V015A,H019K, S030G, NO: 750 N033S, S044D, K053R, A056T, H058A, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408340SEQ ID H019K, S027K, S030G, V031I, N033S, T038S, NO: 751 S044D, K053R,S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V,S086A D_D0408353 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO:752 S027K, S030G, V031I, G032D, N033S, T038S, S044D, K053S, H058A,S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V,S086A D_D0408357 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO:753 S030G, N033S, S044D, S065T, V070I, D071E, N073S, A074T, R080E,L081T, E083H, L085V, S086A D_D0408359 SEQ ID N008K, N011K, P012K, V015A,H019K, S027K, NO: 754 S030G, V031I, G032D, N033S, T038S, S044D, V070I,D071E, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408361 SEQ IDN008K, N011K, P012K, V015A, A016S, S030G, NO: 755 V031I, G032D, N033S,T038S, S044D, K053R, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408366 SEQ ID N008K, N011K, P012K, V015A,A016S, H019K, NO: 756 S027K, S030G, V031I, G032D, N033S, T038S, S044D,K053R, S065T, V070I, D071E, L085V, S086A D_D0408368 SEQ ID N011K, S027K,S030G, V031I, G032D, N033S, NO: 757 T038S, S044D, K053R, A056T, H058A,S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V,S086A D_D0408370 SEQ ID N008K, N011K, P012K, V015A, H019K, S027K, NO:758 S030G, V031I, G032D, N033S, T038S, S044D, S065T, V070I, D071E,N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408372 SEQ IDN011K, V015A, H019K, S027K, S030G, V031I, NO: 759 G032D, N033S, T038S,S044D, K053R, H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408373 SEQ ID N008K, N011K, P012K, V015A,A016S, H019K, NO: 760 S027K, S030G, V031I, G032D, N033S, T038S, S044D,K053R, S065T, V070I, D071E, N073S, A074T, R080E, E083H, L085V D_D0408374SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 761 S027K, S030G,V031I, G032D, N033S, S044D, K053R, S065T, V070I, D071E, N073S, A074T,Q078H, R080E, L081T, E083H, L085V, S086A D_D0408377 SEQ ID N011K, P012K,S027K, S030G, V031I, G032D, NO: 762 N033S, T038S, S044D, K053R, A056T,H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H,L085V, S086A D_D0408381 SEQ ID N008K, N011K, P012K, V015A, A016S, H019K,NO: 763 S027K, S030G, V031I, G032D, N033S, T038S, S044D, S065T, V070I,D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0408382SEQ ID N011K, P012K, H019K, S027K, S030G, V031I, NO: 764 G032D, N033S,T038S, S044D, K053R, S065T, V070I, D071E, N073S, A074T, Q078H, R080E,L081T, E083H, L085V, S086A D_D0408384 SEQ ID N008K, N011K, P012K, V015A,A016S, S027K, NO: 765 S030G, V031I, G032D, N033S, T038S, S044D, S065T,V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H, L085V, S086AD_D0408385 SEQ ID N011K, P012K, H019K, S027K, S030G, V031I, NO: 766G032D, N0335, T038S, S044D, S065T, V070I, D071E, N073S, A074T, Q078H,R080E, L081T, E083H, L085V, S086A D_D0408386 SEQ ID N008K, N011K, P012K,V015A, A016S, H019K, NO: 767 S027K, S030G, V031I, G032D, N033S, S044D,H058A, S065T, V070I, D071E, N073S, A074T, Q078H, R080E, L081T, E083H,L085V, S086A D_D0408387 SEQ ID N008K, N011K, P012K, S027K, S030G, V031I,NO: 768 G032D, N033S, T038S, S044D, K053R, S065T, V070I, D071E, N073S,A074T, Q078H, R080E, L081T, E083H, L085V, S086A D_D0348839 SEQ ID N008K,N011K, P012K, V015A, A016S, H019K, NO: 772 S027K, S030G, V031I, G032D,N033S, T038S, S044D, Q078H, R080E, L081T, E083H, L085V, S086A D_D0347298SEQ ID N008K, N011K, P012K, V015A, A016S, H019K, NO: 771 S027K, S030G,V031I, G032D, N033S, T038S, S044D

Example 10—Identification of Amino Acid Positions Affecting the Functionof PIP-72Aa

The protein sequence alignment of PIP-72Aa (SEQ ID NO: 2) and otheractive family members PIP-72 Da (SEQ ID NO: 10), PIP-72Fa (SEQ ID NO:18), GBP_A3175 (SEQ ID NO: 20) and PIP-72Gb (SEQ ID NO: 32) is shown inFIG. 2. From the alignment, conserved amino acid positions 7, 10, 20,23, 24, 34, 60, 61, 66, 68, 75, 77, 79, 84 were selected for saturationmutagenesis using the oligonucleotide primers indicated in Table 23(QuikChange™ technique, Agilent, 5301 Stevens Creek Blvd, Santa ClaraCalif.). In addition, positions 8, 11, 12, 15, 16, 19, 27, 30, 31, 32,33, 53, 56, 58, 65, 70, 71, 73, 74, 78, 80, 81, 83, 85, 86, which differbetween PIP-72Aa (SEQ ID NO: 2) and PIP-72 Da (SEQ ID NO: 10) weresubjected to saturation mutagenesis using a modification of theQuikChange™ method with Phusion® High Fidelity DNA Polymerase (NewEngland Biolabs®, Cat #M0530L, 240 County Road, Ipswich, Mass.01938-2723), with the oligonucleotide primers indicated in Table 23. Theresulting plasmid DNAs were transformed into E. coli, and protein wasexpressed as described in Example 3. Bioassays were performed on WesternCorn Root Worm as described in Example 4 to determine which genevariants retained insecticidal activity. Table 23 lists the positionsthat were addressed and summarizes the data that was obtained. The aminoacid substitutions that were identified by DNA sequence analysis areshown. For each position, the amino acid substitutions which yieldedPIP-72Aa variants that retained Western Corn Root Worm activity areshown.

TABLE 23 Oligo sequence Identified Active Position Oligo Name identifierSubstitutions Substitutions N7 NNK7_F SEQ ID NO: 190 A, V, C, E, F, G,H, I, A, V MNN7_R SEQ ID NO: 191 K, L, M, P, Q, R, S, T, W, Y N8 N8N_FSEQ ID NO: 192 A, C, D, E, G, H, I, K, A, C, D, E, G, H, I, K, N8N_R SEQID NO: 193 L, M, Q, R, S, T, V, F L, M, Q, R, S, T, V P, W, Y S10NNK10_F SEQ ID NO: 194 A, E, F, G, H, I, K, L, A, E, F, G, H, I, K, L,MNN10_R SEQ ID NO: 195 N, P, Q, R, T, W N, P, Q, R, T, W N11 N11N_F SEQID NO: 196 A, C, D, E, G, H, I, K, A, C, D, E, G, H, I, K, N11N_R SEQ IDNO: 197 L, M, Q, S, T, V, Y, P, M, Q, S, T, V, Y R, W P12 P12N_F SEQ IDNO: 198 A, C, D, E, G, H, K, L, A, C, D, E, G, H, K, L, P12N_R SEQ IDNO: 199 N, Q, R, S, T, V, W, Y, N, Q, R, S, T, V, W, I Y V15 V15N_F SEQID NO: 200 A, C, I, M, R, D, F, G, A, C, I, M, R V15N_R SEQ ID NO: 201H, L, P, Q, S, T, W, Y A16 A16N_F SEQ ID NO: 202 C, D, E, F, G, H, I, K,none A16N_R SEQ ID NO: 203 L, M, N, P, Q, R, S, T, V, W, Y H19 H19N_FSEQ ID NO: 204 A, E, K, L, P, R, S, Y, A, E, K, L, P, R, S, Y H19N_R SEQID NO: 205 C, D, F, G, I, M, N, T, V, W W20 NNK20_F SEQ ID NO: 206 A, T,D, E, G, I, K, L, A, T MNN20_R SEQ ID NO: 207 M, N, P, Q, R, S, V, Y D23NNK23_F SEQ ID NO: 208 A, G, H, K, M, N, Q, S, A, G, H, K, M, N, Q, S,MNN23_R SEQ ID NO: 209 T, V, C, L, P, R, W, Y T, V G24 NNK24_F SEQ IDNO: 210 D, F, A, E, H, I, L, M, D, F MNN24_R SEQ ID NO: 211 N, P, R, S,T, V, W, Y S27 S27N_F SEQ ID NO: 212 A, C, D, E, F, G, H, N, A, C, D, E,F, G, H, N, S27N_R SEQ ID NO: 213 Q, R, T, I, K, L, M, P, Q, R, T W, YS30 S30N_F SEQ ID NO: 214 A, C, D, E, F, G, H, K, A, C, D, E, F, G, H,K, S30N_R SEQ ID NO: 215 L, M, N, P, Q, R, T, V, L, M, N, P, Q, R, T, W,Y V, W, Y V31 V31N_F SEQ ID NO: 216 I, L, A, C, D, E, F, G, I, L V31N_RSEQ ID NO: 217 H, K, N, P, Q, R, S, T, W, Y G32 G32N_F SEQ ID NO: 218 A,D, E, F, H, K, L, M, A, D, E, F, H, K, L, M, G32N_R SEQ ID NO: 219 N, P,Q, R, S, T, V, W, N, P, Q, R, S, T, V, Y W, Y N33 N33N_F SEQ ID NO: 220A, C, D, E, F, G, H, I, A, C, D, E, F, G, H, I, N33N_R SEQ ID NO: 221 K,L, P, Q, R, S, T, V K, L, P, Q, R, S, T, Y, W V, Y G34 NNK34_F SEQ IDNO: 222 E, F, H, K, L, M, N, Q, E, F, H, K, L, M, N, Q, MNN34_R SEQ IDNO: 223 R, S, T, Y, I, P, V, W R, S, T, Y K53 K53N_F SEQ ID NO: 224 A,C, D, E, F, H, I, L, A, C, D, E, F, H, I, L, K53N_R SEQ ID NO: 225 M, N,p, Q, R, S, T, V, M, N, Q, R, S, T, V, Y, G, W Y A56 A56N_F SEQ ID NO:226 G, L, N, P, Q, R, S, T, G, L, N, P, Q, R, S, T A56N_R SEQ ID NO: 227C, D, E, F, H, I, K, Y H58 H58N_F SEQ ID NO: 228 A, D, F, L, M, N, R, W,A, D, F, L, M, N, R, W, H58N_R SEQ ID NO: 229 Y, C, G, I, K, P, S, T, YV Y60 NNK60_F SEQ ID NO: 230 E, F, A, D, G, I, K, L, E, F MNN60_R SEQ IDNO: 231 M, N, P, Q, R, S, T, V Y61 NNK61_F SEQ ID NO: 232 A, C, D, G, I,K, L, N, none MNN61_R SEQ ID NO: 233 P, R, S, T, V, W S65 S65N_F SEQ IDNO: 234 A, C, D, E, F, G, H, I, A, C, D, E, F, G, H, I, S65N_R SEQ IDNO: 235 L, N, T, V, P, R, Y L, N, T, V S66 NNK66_F SEQ ID NO: 236 A, G,C, D, E, I, K, L, A, G MNN66_R SEQ ID NO: 237 M, N, P, R, T, V, W I68NNK68_F SEQ ID NO: 238 D, L, V, A, C, E, F, G, D, L, V MNN68_R SEQ IDNO: 239 H, K, M, N, P, Q, R, T, W, Y V70 V70N_F SEQ ID NO: 240 C, I, A,D, F, G, H, L, C, I V70N_R SEQ ID NO: 241 M, N, P, Q, R, S, T, Y D71D71N_F SEQ ID NO: 242 A, C, G, H, I, L, M, N, A, C, G, H, I, L, M, N,D71N_R SEQ ID NO: 243 S, T, V, Y, F, P, R S, T, V, Y N73 N73N_F SEQ IDNO: 244 A, C, D, F, G, H, I, L, A, C, D, F, G, H, I, L, N73N_R SEQ IDNO: 245 R, S, T, V, Y, P R, S, T, V, Y A74 A74N_F SEQ ID NO: 246 C, D,F, G, H, I, L, N, C, D, F, G, H, I, L, N, A74N_R SEQ ID NO: 247 Q, R, S,T, V, Y, P Q, R, S, T, V, Y V75 NNK75_F SEQ ID NO: 248 C, I, L, A, D, F,G, H, C, I, L MNN75_R SEQ ID NO: 249 K, M, P, Q, R, S, T, W, Y D77NNK77_F SEQ ID NO: 250 Y, A, E, F, G, H, K, L, Y MNN77_R SEQ ID NO: 251M, N, P, Q, R, T, V, W Q78 Q78N_F SEQ ID NO: 252 A, C, D, F, G, H, I, L,A, C, D, F, G, H, I, L, Q78N_R SEQ ID NO: 253 M, N, R, S, T, V, Y, P M,N, R, S, T, V, Y R79 NNK79_F SEQ ID NO: 254 A, C, D, E, F, H, K, L, A,C, D, E, F, H, K, L, MNN79_R SEQ ID NO: 255 N, Q, R, S, T, W, Y, I, N,Q, R, S, T, W, Y P, V R80 R80N_F SEQ ID NO: 256 A, C, D, F, G, H, I, L,A, C, D, F, G, H, I, L, R80N_R SEQ ID NO: 257 N, S, T, V, Y, P N, S, T,V, Y L81 L81N_F SEQ ID NO: 258 A, C, D, F, G, H, I, N, A, C, D, F, G, H,I, N, L81N_R SEQ ID NO: 259 P, R, S, T, V, Y P, R, S, T, V E83 E83N_FSEQ ID NO: 260 A, C, D, F, G, H, I, K, A, C, D, F, G, H, I, K, E83N_RSEQ ID NO: 261 L, N, P, R, S, T, V, Y L, N, P, R, S, T, V, Y P84 NNK84_FSEQ ID NO: 262 A, C, E, I, S, V, W, Y, A, C, E, I, S, V, W, Y MNN84_RSEQ ID NO: 263 F, G, H, L, N, R, T L85 L85N_F SEQ ID NO: 264 C, G, V, A,D, E, F, H, C, G, V L85N_R SEQ ID NO: 265 I, N, P, Q, R, S, T, Y S86S86N_F SEQ ID NO: 266 A, I, T, V, C, D, F, G, A, I, T, V S86N_R SEQ IDNO: 267 H, L, M, N, P, Q, R, Y

Example 11—Identification of Motif 1 and Analysis of Amino AcidPositions Affecting the Function of PIP-72Aa

From the protein sequence alignment of PIP-72Aa (SEQ ID NO: 2) and otheractive family members including PIP-72 Da (SEQ ID NO: 10), PIP-72Fa (SEQID NO: 18), GBP_A3175 (SEQ ID NO: 20) and PIP-72Gb (SEQ ID NO: 32) shownin FIG. 2, the conserved Motif 1 was identified as potentially importantfor function (underlined in FIG. 2 relative to PIP-72Aa (SEQ ID NO: 2).Each position within Motif 1 (37, 38, 39, 40, 41, 42, 43, 44, 45, 46,47, 48, 49, 50 and 51) was subjected to saturation mutagenesis using theoligonucleotide primers indicated in Table 24. The resulting plasmidDNAs were transformed into E. coli and protein was expressed asdescribed in Example 3. Bioassays were performed on Western Corn RootWorm as described in Example 4 to determine which gene variants retainedinsecticidal activity. Table 24 lists the positions that weremutagenized and summarizes the data that was obtained. For eachposition, the amino acid substitutions which yielded PIP-72A polypeptidevariants that retained Western Corn Root Worm activity are shown. Also,the amino acid substitutions associated with soluble protein expressionin E. coli are noted.

TABLE 24 Res- Oligo Sequence Identified Active Soluble idue Nameidentifier Substitutions Substitutions Expression E37 37nnk_F SEQ ID A,C, D, E, F, A, C, D, F, G, A, C, D, E, NO: 268 G, I, K, L, M, I, K, L,M, N, F, G, I, K, N, S, T, V, H S, T, V L, M, M, S P, R, W, Y T, V, H,R, W, Y T38 T38N_F SEQ ID A, C, D, E, F, A, C, D, E, F, A, C, D, E, NO:269 G, H, I, L, M, G, H, I, L, M, F, G, H, I, T38N_R SEQ ID N, Q, R, S,V, N, Q, R, S, V, L, M, N, Q, NO: 270 W, Y, K, P W, Y R, S, V, W, Y W3939nnk_F SEQ ID F, A, C, D, E, F F, W, Y NO: 271 G, H, I, K, L, M, N, P,Q, R, S, T, V, Y D40 40nnk_F SEQ ID A, C, E, F, G, A, C, E, F, G, A, C,D, E, NO: 272 H, I, K, L, M, H, I, K, L, M, F, G, H, I, N, Q, R, S, T,N, Q, R, S, T, K, L, M, N, V, W, Y, P V, W, Y Q, R, S, T, V, W, Y, P R4141nnk_F SEQ ID A, C, D, E, F, none F, I, L, N, NO: 273 G, I, K, L, M, VN, P, Q, S, T, V, W, Y S42 42nnk_F SEQ ID A, C, D, E, F, A, C, D, E, F,A, C, D, E, NO: 274 G, I, K, L, M, G, I, K, L, M, F, G, I, K, N, Q, R,S, T, N, Q, R, T, V, L, M, N, Q, V, W, Y, P W, Y R, S, T, V, W, Y, P D4343nnk_F SEQ ID A, C, E, F, G, none NO: 275 H, I, K, L, N, P, Q, R, S, T,V, W, Y S44 S44N_F SEQ ID A, D, E, G, L, A, D, E, G, L, A, D, E, G, NO:276 M, N, P, Q, T, M, N, P, Q, T, L, M, N, P, S44N_R SEQ ID V, Y, H, I,K, V, Y Q, T, V, Y, NO: 277 R, W H, I, K, R, W R45 45nnk_F SEQ ID K, R,S, A, C, K, S K, R, L, M, NO: 278 D, E, F, G, H, Q, S, Y L, M, N, P, Q,T, V, W, Y G46 46nnk_F SEQ ID A, Q, C, D, E, A, Q A, G, Q NO: 279 F, H,I, K, L, M, N, P, R, S, T, V, W, Y F47 47nnk_F SEQ ID C, V, Y, A, D, C,V, Y C, F, V, Y, NO: 280 E, G, H, I, K, H, L, M L, M, N, P, Q R, S, T, WV48 48nnk_F SEQ ID I, L, A, C, D, I, L I, L, V NO: 281 E, F, G, H, K, M,N, P, Q, R, S, W L49 49nnk_F SEQ ID C, F, M, R, Y, C, F, M, R, Y C, F,L, M, NO: 282 A, D, E, G, H, R, Y I, K, N, P, Q, S, T, V, W S50 50nnk_FSEQ ID A, C, D, I, M, A, C, D, I, M, A, C, D, I, NO: 283 P, Q, S, T, V,P, Q, T, V M, Q, S, T, F, G, H, K, L, V, F, H, L, N, R, W, Y N, R, W, YL51 51nnk_F SEQ ID A, C, M, V, D, A, C, M, V A, C, L, M, NO: 284 E, F,G, H, K, V N, P, Q, R, S, T, W, Y

Example 12—PIP-72Aa Variants with Multiple Amino Acid Substitutions inMotif 1

PIP-72Aa variants with multiple selected amino acid substitutions inMotif 1 were generated by the QuikChange™ technique (Agilent, SantaClara Calif.) using a combination of the oligonucleotide primersindicated in Table 25. The resulting plasmid DNAs were transformed intoE. coli, and protein was expressed as described in Example 3. Bioassayswere performed on Western Corn Root Worm as described in Example 4 todetermine which gene variants retained insecticidal activity. DNAsequencing was performed on the active gene variants. The nucleotide andamino acid sequences of active variants are listed in Table 26. Out of78 variants screened, 20 had detectable Western Corn Root Worm activity.The active variants contained between 2 and 7 amino acid substitutionsrelative to PIP-72Aa (SEQ ID NO: 2). Table 27 shows the amino acidsubstitutions of the resulting active PIP-72 polypeptide variantscompared to PIP-72Aa (SEQ ID NO: 2).

TABLE 25 Sequence Sequence primer identifier primer identifierCombiMM1:1_T SEQ ID NO: 773 CombiMM1:17_T SEQ ID NO: 789 CombiMM1:2_TSEQ ID NO: 774 CombiMM1:18_T SEQ ID NO: 790 CombiMM1:3_T SEQ ID NO: 775CombiMM1:19_T SEQ ID NO: 791 CombiMM1:4_T SEQ ID NO: 776 CombiMM1:20_TSEQ ID NO: 792 CombiMM1:5_T SEQ ID NO: 777 CombiMM1:21_T SEQ ID NO: 793CombiMM1:6_T SEQ ID NO: 778 CombiMM1:22_T SEQ ID NO: 794 CombiMM1:7_TSEQ ID NO: 779 CombiMM1:23_T SEQ ID NO: 795 CombiMM1:8_T SEQ ID NO: 780CombiMM1:24_T SEQ ID NO: 796 CombiMM1:9_T SEQ ID NO: 781 CombiMM1:25_TSEQ ID NO: 797 CombiMM1:10_T SEQ ID NO: 782 CombiMM1:26_T SEQ ID NO: 798CombiMM1:11_T SEQ ID NO: 783 CombiMM1:27_T SEQ ID NO: 799 CombiMM1:12_TSEQ ID NO: 784 CombiMM1:28_T SEQ ID NO: 800 CombiMM1:13_T SEQ ID NO: 785CombiMM1:29_T SEQ ID NO: 801 CombiMM1:14_T SEQ ID NO: 786 CombiMM1:30_TSEQ ID NO: 802 CombiMM1:15_T SEQ ID NO: 787 CombiMM1:31_T SEQ ID NO: 803CombiMM1:16_T SEQ ID NO: 788 CombiMM1:32_T SEQ ID NO: 804

TABLE 26 % ID PIP- Clone 72Aa Designation Polynucleotide Polypeptide95.35% D_D0416473 SEQ ID NO: 805 SEQ ID NO: 825 95.35% D_D0416474 SEQ IDNO: 806 SEQ ID NO: 826 96.51% D_D0416483 SEQ ID NO: 807 SEQ ID NO: 82794.19% D_D0416488 SEQ ID NO: 808 SEQ ID NO: 828 95.35% D_D0416490 SEQ IDNO: 809 SEQ ID NO: 829 97.67% D_D0416492 SEQ ID NO: 810 SEQ ID NO: 83097.67% D_D0416493 SEQ ID NO: 811 SEQ ID NO: 831 95.35% D_D0416502 SEQ IDNO: 812 SEQ ID NO: 832 96.51% D_D0416506 SEQ ID NO: 813 SEQ ID NO: 83394.19% D_D0416507 SEQ ID NO: 814 SEQ ID NO: 834 94.19% D_D0416514 SEQ IDNO: 815 SEQ ID NO: 835 94.19% D_D0416520 SEQ ID NO: 816 SEQ ID NO: 83691.86% D_D0416521 SEQ ID NO: 817 SEQ ID NO: 837 96.51% D_D0416527 SEQ IDNO: 818 SEQ ID NO: 838 94.19% D_D0416528 SEQ ID NO: 819 SEQ ID NO: 83995.35% D_D0416542 SEQ ID NO: 820 SEQ ID NO: 840 94.19% D_D0416543 SEQ IDNO: 821 SEQ ID NO: 841 95.35% D_D0416551 SEQ ID NO: 822 SEQ ID NO: 84291.86% D_D0416552 SEQ ID NO: 823 SEQ ID NO: 843 95.35% D_D0416555 SEQ IDNO: 824 SEQ ID NO: 844

TABLE 27 Clone des. polypeptide Subtitutions relative to SEQ ID NO: 2D_D0416473 SEQ ID NO: 825 E037V, T0385, D040E, S044A D_D0416474 SEQ IDNO: 826 E037V, D040G, S044A, S050G D_D0416483 SEQ ID NO: 827 L049M,S050C, L051M D_D0416488 SEQ ID NO: 828 T038G, S044A, L049M, S050C, L051MD_D0416490 SEQ ID NO: 829 D040G, S042T, S044N, S050C D_D0416492 SEQ IDNO: 830 S050C, L051M D_D0416493 SEQ ID NO: 831 S044N, L049M D_D0416502SEQ ID NO: 832 E037V, T0385, D040A, S050C D_D0416506 SEQ ID NO: 833E037C, T038G, S050C D_D0416507 SEQ ID NO: 834 E037V, T038G, S044A,L049M, S050A D_D0416514 SEQ ID NO: 835 E037V, T038G, D040E, S044N, S050CD_D0416520 SEQ ID NO: 836 E037C, T038G, S042T, S044N, L049M D_D0416521SEQ ID NO: 837 E037V, T0385, D040G, S044A, V0481, S050C, L051MD_D0416527 SEQ ID NO: 838 T038G, S042T, S050C D_D0416528 SEQ ID NO: 839E037D, D040A, S042T, L049M, S050C D_D0416542 SEQ ID NO: 840 E037C,D040G, L049M, S050A D_D0416543 SEQ ID NO: 841 E037C, T0385, D040E,S042T, S050C D_D0416551 SEQ ID NO: 842 E037V, T0385, D040A, S042ND_D0416552 SEQ ID NO: 843 E037V, D040E, S042T, S044N, L049M, S050C,L051M D_D0416555 SEQ ID NO: 844 E037D, T0385, S044A, S050C

Example 13—Identification of Additional Amino Acid Positions Affectingthe Function of PIP-72Aa

The remaining amino acid positions 2, 3, 4, 5, 6, 9, 13, 14, 17, 18, 21,22, 25, 26, 28, 29, 35, 36, 52, 54, 55, 57, 59, 62, 63, 64, 67, 69, 72,76, 82 were subjected to saturation mutagenesis as described in Example11, using the oligonucleotide primers indicated in Table 27. Theresulting plasmid DNAs were transformed into E. coli, and protein wasexpressed as described in Example 3. Bioassays were performed on WesternCorn Root Worm as described in Example 4 to determine which genevariants retained insecticidal activity. Table 28 lists the positionsthat were mutagenized and summarizes the amino acid substitutionsidentified by DNA sequencing and the amino acid substitutions whichyielded PIP-72Aa variants that retained Western Corn Root Worm activityare shown.

TABLE 28 OLIGO SEQUENCE IDENTIFIED ACTIVE POSITION NAME IDENTIFIERSUBSTITUTIONS SUBSTITUTIONS G2 NNK2_HM SEQ ID NO: 865 A, C, D, E, I, K,L, N, A, C, D, E, I, K, L, N, R, S, T, V, W, Y R, S, T, V, W, Y I3NNK3_HM SEQ ID NO: 866 A, C, D, E, F, G, H, L, W N, P, Q, R, S, V, W, YT4 NNK4_HM SEQ ID NO: 867 A, D, E, F, G, H, I, K, A, D, E, H, I, K, L,R, L, P, R, S, V, W, Y S, V, W, Y V5 NNK5_HM SEQ ID NO: 868 A, C, E, G,H, I, K, L, A, C, G, H, I, Y N, Q, R, S, T, W, Y T6 NNK6_HM SEQ ID NO:869 A, C, D, E, F, G, H, I, A, C, F, G, H, I, K, M, K, L, M, N, P, Q, R,S, P, Q, R, S, W, Y V, W, Y S9 NNK9 SEQ ID NO: 889 A, C, D, E, F, G, H,I, A, C, G, T MNN9 SEQ ID NO: 890 K, L, M, P, Q, R, T, V, W, Y I13NNK13_HM SEQ ID NO: 870 A, C, D, F, G, H, K, L, N, Q, V M, N, P, Q, R,S, T, V, W, Y E14 NNK14_HM SEQ ID NO: 871 A, C, D, F, G, H, K, L, A, C,F, H, K, Q M, N, P, Q, R, S, T, V, W, Y I17 NNK17_HM SEQ ID NO: 872 A,C, D, E, F, G, H, K, E, V L, M, N, P, Q, R, S, T, V, W, Y N18 NNK18_HMSEQ ID NO: 873 A, C, D, E, F, G, H, I, S K, L, M, Q, R, S, T, V, Y G21NNK21_HM SEQ ID NO: 874 A, D, F, I, L, M, P, R, NONE S, T, V, W S22NNK22_HM SEQ ID NO: 875 A, C, D, E, F, G, H, I, A, D, F, G, H, I, K, L,K, L, M, N, P, Q, R, T, M, N, P, Q, R, T, V, V, W, Y Y D25 NNK25_HM SEQID NO: 876 A, C, E, F, G, H, L, N, A, E, F, N, Q P, Q, R, S, V, W T26NNK26_HM SEQ ID NO: 877 A, C, D, E, F, G, H, I, E, P K, L, M, N, P, Q,R, S, V, W, Y F28 NNK28 SEQ ID NO: 891 A, C, D, E, G, H, I, K, P, W, YMNN28 SEQ ID NO: 892 L, M, N, P, Q, R, S, T, V, W, Y F29 NNK29 SEQ IDNO: 893 A, C, D, E, G, H, I, K, A, C, I, L, Q, R, W, Y MNN29 SEQ ID NO:894 L, M, N, P, S, T, V, W, Y K35 NNK35 SEQ ID NO: 895 A, C, D, G, H, I,L, M, A, C, D, G, H, I, L, M, MNN35 SEQ ID NO: 896 N, P, Q, R, S, T, V,W, N, Q, R, S, T, V Y Q36 NNK36_HM SEQ ID NO: 878 A, C, E, G, H, I, K,L, A, C, E, G, H, I, K, L, M, N, P, R, S, T, V, W N, P, R, S, T, V Y K52NNK52 SEQ ID NO: 897 A, C, D, E, F, G, H, I, C, F, H, I, L, M, N, R,MNN52 SEQ ID NO: 898 L, M, N, P, R, S, T, V, S, T, W, Y W, Y N54NNK54_HM SEQ ID NO: 879 A, C, D, E, F, G, I, K, C, D, E, F, G, K, M, Q,L, M, P, Q, R, S, T, V R, S, W W, Y G55 NNK55_HM SEQ ID NO: 880 A, C, D,E, F, H, I, K, NONE L, M, N, P, Q, R, S, T, V, W, Y Q57 NNK57_HM SEQ IDNO: 881 A, C, D, E, F, G, H, I, E, L, M, S, T K, L, M, P, R, S, T, V, WP59 NNK59_HM SEQ ID NO: 882 A, C, D, E, F, G, H, I, NONE K, L, M, N, Q,R, S, T, V, W, Y V62 NNK62_HM SEQ ID NO: 883 A, C, D, E, F, G, H, K,NONE L, M, N, P, Q, R, S, T, W, Y Q63 NNK63_HM SEQ ID NO: 884 A, C, D,E, F, G, H, I, C, G, I, L, M, N, T, V, K, L, M, N, P, R, S, T, Y V, W, YA64 NNK64_HM SEQ ID NO: 885 C, D, E, F, G, H, I, K, F, G, H, R, S, Y L,M, N, P, Q, R, S, T, V, W, Y K67 NNK67 SEQ ID NO: 899 A, C, D, F, G, H,I, L, A, C, D, F, H, I, L, M, MNN67 SEQ ID NO: 900 M, N, P, Q, R, S, T,V, N, Q, R, S, T, V, W, W, Y Y E69 NNK69_HM SEQ ID NO: 886 A, C, D, F,G, H, I, K, A, C, D, F, H, I, L, M, L, M, N, P, Q, R, S, T, Q, R, S, T,V, Y V, Y N72 NNK72_HM SEQ ID NO: 887 A, C, D, E, F, G, I, K, A, C, D,E, G, K, M, P, L, M, P, Q, R, S, T, V, Q, R, S, T, V, W W, Y K76 NNK76SEQ ID NO: 901 A, C, D, E, F, G, H, I, A, C, F, H, I, L, Q, R, MNN76 SEQID NO: 902 L, N, P, Q, R, S, T, V, S, T, V, W, Y W, Y I82 NNK82_HM SEQID NO: 888 A, D, E, F, G, H, K, L, A, L, M, R, V M, N, P, Q, R, S, T, V,W, Y

Example 14—PIP-72Aa Variants with Improved Activity Against Western CornRootworm

To create variants of PIP-72Aa (SEQ ID NO: 2) with increased Westerncorn rootworm activity, libraries were generated by synthetic DNAshuffling (Ness et al, 2002, Nature Biotechnology 20, 1251-5) ofPIP-72Aa (SEQ ID NO: 1) and by site-directed mutagenesis (QuikChange™Lightning technique or QuikChange™ II technique, Agilent, Santa ClaraCalif.). Twelve PIP-72 variant polypeptides with increased specificactivity against Western corn rootworm were identified. The PIP-72variant polypeptides A5, A5:10E, A5:10T, A5:10V, A5:10A, A5:10L,A5:10E/78H, A5:8M, A5:71H/83F, A5:4S/54Q/78H, A5:71 H and 3_68 exhibitedbetween about 1.2 and 2-fold improved insecticidal activity againstWestern corn rootworm compared to PIP-72Aa (SEQ ID NO: 2). Table 29shows the amino acid substitutions compared to PIP-72Aa (SEQ ID NO: 2)for each of the PIP-72 variant polypeptides, the corresponding aminoacid sequence identifier, and the corresponding nucleic acid sequenceidentifier. Amino acid substitutions compared to A5 (SEQ ID NO: **) arebolded.

TABLE 29 a.a. substitutions compared Polypeptide Polynucleotide Variantto SEQ ID NO: 2 sequence sequence 72Aa SEQ ID SEQ ID NO: 1 NO: 2 Combi3_S10E, L49M, E83Y SEQ ID SEQ ID NO: 915 68 NO: 903 A5 S30G, V31I, G32D,N33S, SEQ ID SEQ ID NO: 916 T38S, K53R, H57A NO: 904 A5:10E S10E, S30G,V31I, SEQ ID SEQ ID NO: 917 G32D, N33S, NO: 905 T38S, K53R, H57A A5:10TS10T, S30G, V31I, G32D, SEQ ID SEQ ID NO: 918 N33S, T38S, K53R, H57A NO:906 A5:10V S10V, S30G, V31I, G32D, SEQ ID SEQ ID NO: 919 N33S, T38S,K53R, H57A NO: 907 A5:10A S10A, S30G, V31I, G32D, SEQ ID SEQ ID NO: 920N33S, T38S, K53R, H57A NO: 908 A5:10L S10L, S30G, V31I, G32D, SEQ ID SEQID NO: 921 N33S, T38S, K53R, H57A NO: 909 A5:10E/ S10E, S30G, V31I,G32D, SEQ ID SEQ ID NO: 922 78H N33S, T38S, K53R, NO: 910 H57A, Q78HA5:8M N8M, S30G, V31I, SEQ ID SEQ ID NO: 923 G32D, N33S, NO: 911 T38S,K53R, H57A A5:71H/ S30G, V31I, G32D, N33S, SEQ ID SEQ ID NO: 924 83FT38S, K53R, NO: 912 H57A, D71H, E83F A5:4S/ S30G, V31I, G32D, N33S, SEQID SEQ ID NO: 925 54Q/78H T38S, K53R, N54Q, NO: 913 H57A, Q78H A5:71HS30G, V31I, G32D, N33S, SEQ ID SEQ ID NO: 926 T38S, K53R, H57A, D71H NO:914

To determine Western corn rootworm activity, test proteins were assayedas described in Example 4. A 0-3 numerical scoring system based on thesize and mortality was used. If no response or normal growth was seen,Score 0 was given. When the growth was somewhat retarded without anymortality, it was Score 1. Score 2 meant partial death (multiple insectswere used in each well) and strong growth inhibition. Score 3 indicatedthe complete mortality. Each treatment was repeated 6 times for possiblehighest score of 18. In this scoring system, Score 9 with 6 repeats ofone treatment means the 50% response (9 out of 18) of the treatment andcalled ILC50 (growth Inhibition and Lethal Concentration for 50%response). Activity data is shown in Table 30. Converted ILC50 numbersrepresent averages of between 2 and 20 experiments.

TABLE 30 con- con- fold con- verted verted Im- verted lower upper prove-Variant ILC50 range range ment PIP-72Aa 56.68 48.32 66.69 1 n = 18 SEQID NO: 2 3_68 48.86 40.78 58.22 1.16 n = 4 SEQ ID NO: 903 A5 36.68 30.7643.24 1.55 n = 20 SEQ ID NO: 904 A5:10E 33.96 27.78 41.01 1.67 n = 10SEQ ID NO: 905 A5:10T 30.54 23.78 38.48 1.86 n = 4 SEQ ID NO: 906 A5:10V31.35 25.69 37.87 1.81 n = 4 SEQ ID NO: 907 A5:10A 42.38 35.61 50.271.34 n = 2 SEQ ID NO: 908 A5:10L 39.05 31.87 47.78 1.45 n = 2 SEQ ID NO:909 A5:10E/78H 36.04 28.64 44.66 1.57 n = 10 SEQ ID NO: 910 A5:8M 25.0219.34 30.90 2.27 n = 6 SEQ ID NO: 911 A5:71H/83F 32.24 26.72 38.44 1.78n = 4 SEQ ID NO: 912 A5:4S/54Q/78H 29.01 23.42 35.20 1.95 n = 4 SEQ IDNO: 913 A5:71H 26.82 20.57 33.75 2.1 n = 2 SEQ ID NO: 914

The above description of various illustrated embodiments of thedisclosure is not intended to be exhaustive or to limit the scope to theprecise form disclosed. While specific embodiments of and examples aredescribed herein for illustrative purposes, various equivalentmodifications are possible within the scope of the disclosure, as thoseskilled in the relevant art will recognize. The teachings providedherein can be applied to other purposes, other than the examplesdescribed above. Numerous modifications and variations are possible inlight of the above teachings and, therefore, are within the scope of theappended claims.

These and other changes may be made in light of the above detaileddescription. In general, in the following claims, the terms used shouldnot be construed to limit the scope to the specific embodimentsdisclosed in the specification and the claims.

The entire disclosure of each document cited (including patents, patentapplications, journal articles, abstracts, manuals, books or otherdisclosures) in the Background, Detailed Description, and Examples isherein incorporated by reference in their entireties.

Efforts have been made to ensure accuracy with respect to the numbersused (e.g. amounts, temperature, concentrations, etc.) but someexperimental errors and deviations should be allowed for.

That which is claimed:
 1. A DNA construct comprising a nucleic acidmolecule, encoding a PIP-72 polypeptide having insecticidal activityagainst Western corn rootworm (Diabrotica virgifera virgifera), operablylinked to a heterologous regulatory element, wherein the encoded PIP-72polypeptide comprises an amino acid sequence having at least 80%sequence identity to SEQ ID NO:
 2. 2. The DNA construct of claim 1,wherein the encoded PIP-72 polypeptide comprises 1 to 17 amino acidsubstitutions at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52,53, 54, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72,73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 or 86, compared tothe corresponding amino acid of SEQ ID NO:
 2. 3. The DNA construct ofclaim 1, wherein the encoded PIP-72 polypeptide comprises 1 to 17 aminoacid substitutions at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,14, 15, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33,34, 35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 56, 58, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76,77, 78, 79, 80, 81, 82, 83, 84, 85 or 86 compared to the correspondingamino acid of SEQ ID NO:
 2. 4. The DNA construct of claim 1, wherein theencoded PI P-72 polypeptide comprises an amino acid sequence of theformula: (SEQ ID NO: 846)Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala1               5                   10                  15Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa            20                  25                  30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa        35                  40                  45Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Gln Xaa Pro Xaa Tyr Val Xaa Xaa    50                  55                  60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65                  70                  75                  80Xaa Xaa Xaa Xaa Xaa Xaa                 85 

wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu,Asn, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 3 is Ile or Trp;Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser,Val, Trp or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile orTyr; Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, Ile, Lys, Met,Pro, Gln, Arg, Ser, Trp or Tyr; Xaa at position 7 is Asn, Ala or Val;Xaa at position 8 is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu,Met, Gln, Arg, Ser, Thr or Val; Xaa at position 9 is Ser, Ala, Cys, Glyor Thr; Xaa at position 10 is Ser, Ala, Glu, Phe, Gly, His, Ile, Lys,Leu, Asn, Pro, Gln, Arg, Thr or Trp; Xaa at position 11 is Asn, Ala,Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val or Tyr;Xaa at position 12 is Pro, Ala, Cys, Asp, Glu, Gly, His, Lys, Leu, Asn,Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 13 is Ile, Asn, Glnor Val; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys or Gln; Xaaat position 15 is Val, Ala, Cys, Ile, Met or Arg; Xaa at position 17 isIle, Glu or Val; Xaa at position 18 is Asn or Ser; Xaa at position 19 isHis, Ala, Glu, Lys, Leu, Pro, Arg, Ser or Tyr; Xaa at position 20 isTrp, Ala or Thr; Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr or Val; Xaa atposition 24 is Gly, Asp or Phe; Xaa at position 25 is Asp, Ala, Glu,Phe, Asn or Gln; Xaa at position 26 is Thr, Glu or Pro; Xaa at position27 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, Gln, Arg or Thr; Xaaat position 28 is Phe, Pro, Trp or Tyr; Xaa at position 29 is Phe, Ala,Cys, Ile, Leu, Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Ala,Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp or Tyr; Xaa at position 31 is Val, Ile or Leu; Xaa at position32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp or Tyr; Xaa at position 33 is Asn, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val or Tyr;Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr or Tyr; Xaa at position 35 is Lys, Ala, Cys, Asp, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa at position 36 isGln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr orVal; Xaa at position 37 is Glu, Ala, Cys, Asp, Phe, Gly, Ile, Lys, Leu,Met, Asn, Ser, Thr or Val; Xaa at position 38 is Thr, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr;Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; Xaa at position 42 is Ser, Ala, Cys, Asp, Glu, Phe, Gly,Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position44 is Ser, Ala, Asp, Glu, Gly, Leu, Met, Asn, Pro, Gln, Thr, Val or Tyr;Xaa at position 45 is Arg, Lys or Ser; Xaa at position 46 is Gly, Ala orGln; Xaa at position 47 is Phe, Cys, Val or Tyr; Xaa at position 48 isVal, Ile or Leu; Xaa at position 49 is Leu, Cys, Phe, Met, Arg or Tyr;Xaa at position 50 is Ser, Ala, Cys, Asp, Ile, Met, Pro, Gln, Thr orVal; Xaa at position 51 is Leu, Ala, Cys, Met or Val; Xaa at position 52is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Trp or Tyr;Xaa at position 53 is Lys, Ala, Cys, Asp, Glu, Phe, His, Ile, Leu, Met,Asn, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 54 is Asn, Cys,Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position 56is Ala, Gly, Leu, Asn, Pro, Gln, Arg, Ser or Thr; Xaa at position 57 isGln, Glu, Leu, Met, Ser or Thr; Xaa at position 58 is His, Ala, Asp,Phe, Leu, Met, Asn, Arg, Trp or Tyr; Xaa at position 60 is Tyr, Glu orPhe; Xaa at position 63 is Gln, Cys, Gly, Ile, Leu, Met, Asn, Thr, Valor Tyr; Xaa at position 64 is Ala, Phe, Gly, His, Arg, Ser or Tyr; Xaaat position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn,Thr or Val; Xaa at position 66 is Ser, Ala or Gly; Xaa at position 67 isLys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu or Val; Xaa atposition 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Cys or Ile; Xaa atposition 71 is Asp, Ala, Cys, Gly, His, Ile, Leu, Met, Asn, Ser, Thr,Val or Tyr; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys,Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 73 is Asn,Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Ser, Thr, Val or Tyr; Xaa atposition 74 is Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 75 is Val, Cys, Ile or Leu; Xaa atposition 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 77 is Asp Tyr; Xaa at position 78 isGln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr,Val or Tyr; Xaa at position 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe,His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp or Tyr; Xaa at position 80is Arg, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Val orTyr; Xaa at position 81 is Leu, Ala, Cys, Asp, Phe, Gly, His, Ile, Asn,Pro, Arg, Ser, Thr or Val; Xaa at position 82 is Ile, Ala, Leu, Met, Argor Val; Xaa at position 83 is Glu, Ala, Cys, Asp, Phe, Gly, His, Ile,Lys, Leu, Asn, Pro, Arg, Ser, Thr, Val or Tyr; Xaa at position 84 isPro, Ala, Cys, Glu, Ile, Ser, Val, Trp or Tyr; Xaa at position 85 isLeu, Cys, Gly or Val; and Xaa at position 86 is Ser, Ala, Ile, Thr orVal.
 5. The DNA construct of claim 1, wherein the encoded PIP-72polypeptide comprises an amino acid sequence of the formula:SEQ ID NO: 849Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa1               5                   10                  15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa            20                  25                  30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa        35                  40                  45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa    50                  55                  60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65                  70                  75                  80Xaa Xaa Xaa Xaa Xaa Xaa                 85

wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu,Asn, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 3 is Ile, Leu, Valor Trp; Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu,Arg, Ser, Val, Trp or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His,Ile, Leu or Tyr; Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, Ile,Lys, Met, Pro, Gln, Arg, Ser, Trp or Tyr; Xaa at position 7 is Asn, Alaor Val; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thr, Ala,Cys, Asp, Glu, His, Ile, Leu, Met or Val; Xaa at position 9 is Ser, Ala,Cys, Gly or Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, Ser,Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, Met, Val or Tyr; Xaa at position12 is Pro, Thr, Lys, Ser, Arg, Ala, Cys, Asp, Glu, Gly, His, Leu, Asn,Gln, Arg, Val, Trp or Tyr; Xaa at position 13 is Ile, Asn, Gln, Leu orVal; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, Asp or Gln; Xaaat position 15 is Val, Ala, Ile, Leu, Cys, Met or Arg; Xaa at position16 is Ala or Ser; Xaa at position 17 is Ile, Glu, Leu or Val; Xaa atposition 18 is Asn, Gln, Thr or Ser; Xaa at position 19 is His, Lys,Ala, Arg, Glu, Leu, Pro, Ser or Tyr; Xaa at position 20 is Trp, Ala orThr; Xaa at position 21 is Gly, Arg or Lys; Xaa at position 22 is Ser,Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val or Tyr; Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn,Gln, Ser, Thr or Val; Xaa at position 24 is Gly, Asp or Phe; Xaa atposition 25 is Asp, Ala, Glu, Phe, Asn or Gln; Xaa at position 26 isThr, Glu, Asp, Ser or Pro; Xaa at position 27 is Ser, Thr, Lys, Arg,Ala, Cys, Asp, Glu, Phe, Gly, His, Asn or Gln; Xaa at position 28 isPhe, Tyr, Pro or Trp; Xaa at position 29 is Phe, Ala, Cys, Ile, Leu,Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Gly, Lys, Thr, Arg,Ala, Cys, Asp, Glu, Phe, His, Leu, Met, Asn, Pro, Gln, Val, Trp or Tyr;Xaa at position 31 is Val, Ile, Met or Leu; Xaa at position 32 is Gly,Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 33 is Asn, Ser, Gln, Pro, Thr, Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Arg, Val or Tyr; Xaa atposition 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr or Tyr; Xaa at position 35 is Lys, Glu, Ala, Cys, Asp, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa at position 36 isGln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr orVal; Xaa at position 37 is Glu, Asp, Ala, Cys, Phe, Gly, Ile, Lys, Leu,Met, Asn, Ser, Thr or Val; Xaa at position 38 is Thr, Ser, Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Val, Trp or Tyr;Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; Xaa at position 42 is Ser, Asn, Thr, Ala, Cys, Asp, Glu,Phe, Gly, Ile, Lys, Leu, Met, Arg, Val, Trp, Tyr or Gln; Xaa at position44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, Ala, Gly, Leu, Met, Asn, Pro,Gln, Val, Tyr or Val; Xaa at position 45 is Arg, Lys or Ser; Xaa atposition 46 is Gly, Ala or Gln; Xaa at position 47 is Phe, Tyr Cys, Valor Trp; Xaa at position 48 is Leu, Met, Ile, Cys, Phe, Met, Arg, Tyr orVal; Xaa at position 49 is Leu, Met, Ile or Val; Xaa at position 50 isSer, Ala, Tyr, Cys, Asp, Ile, Met, Pro, Gln, Val or Thr; Xaa at position51 is Leu, Val, Ala, Cys, Met or Ile; Xaa at position 52 is Lys, Cys,Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Gln, Trp or Tyr; Xaa atposition 53 is Lys, Arg, Met, Leu, Ile, Ala, Cys, Asp, Glu, Phe, His,Asn, Gln, Ser, Thr, Tyr or Val; Xaa at position 54 is Asn, Cys, Asp,Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position 55 isGly, Ser or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, Gly, Leu,Pro, Arg or Asn; Xaa at position 57 is Gln, Glu, Leu, Met, Ser, Val,Ala, Asn, Ile or Thr; Xaa at position 58 is His, Ala, Lys, Asp, Phe,Leu, Met, Asn, Arg, Trp, Tyr or Thr; Xaa at position 59 is Pro, Thr orSer; Xaa at position 60 is Tyr, Glu or Phe; Xaa at position 62 is Val,Ile or Leu; Xaa at position 63 is Gln, Ser, Cys, Gly, Ile, Leu, Met,Asn, Thr, Val or Tyr; Xaa at position 64 is Ala, Gln, Asn, Phe, Gly,His, Arg, Ser or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Leu, Asn, Val or Thr; Xaa at position 66 is Ser, Alaor Gly; Xaa at position 67 is Lys, Gln, Asn or Arg; Xaa at position 67is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu or Val; Xaa atposition 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Ile, Cys or Leu; Xaa atposition 71 is Asp, Glu, Tyr, Ala, Cys, Gly, His, Ile, Leu, Met, Asn,Ser, Thr, Val or Trp; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu,Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, His or Trp; Xaa at position73 is Asn, Ser, Asp, Gln, Thr, Ala, Cys, Phe, Gly, His, Ile, Leu, Val,Tyr or Glu; Xaa at position 74 is Ala, Thr, Met, Ile, Lys, Ser, Leu,Val, Cys, Asp, Phe, Gly, His, Asn, Gln, Tyr or Arg; Xaa at position 75is Val, Cys, Ile or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His,Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 77 is AspTyr; Xaa at position 78 is Gln, His, Ser, Asn, Ala, Cys, Asp, Phe, Gly,Ile, Leu, Met, Asn, Arg, Val, Tyr or Thr; Xaa at position 79 is Gly,Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Trp or Tyr; Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, Ala, Cys,Phe, Gly, His, Ile, Leu, Ser, Thr, Val, Tyr or Asn; Xaa at position 81is Leu, Pro, Thr, Ile, Val, Ala, Cys, Asp, Phe, Gly, His or Ser; Xaa atposition 82 is Ile, Ala, Leu, Met, Arg and Val; Xaa at position 83 isGlu, His, Asn, Leu, Gln, Ile, Ala, Cys, Asp, Phe, Gly, Lys, Pro, Arg,Ser, Thr, Tyr or Val; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile,Ser, Val, Trp or Tyr; Xaa at position 85 is Leu, Val, Cys, Gly or Ala;and Xaa at position 86 is Ser, Ala, Tyr, Asn, Ile, Val or Thr.
 6. TheDNA construct of claim 1, wherein the encoded PIP-72 polypeptidecomprises an amino acid motif as represented by positions 37-51 of SEQID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848 or SEQ ID NO:
 849. 7. A DNAconstruct comprising a chimeric nucleic acid molecule encoding achimeric PIP-72 polypeptide comprising at least a first moietycomprising a portion of a first PIP-72 polypeptide encoded by thenucleic acid molecule of claim 1 and a second moiety comprising acomplementary portion of a second PIP-72 polypeptide encoded by thenucleic acid molecule of claim 1, wherein the first PIP-72 polypeptideand second PIP-72 polypeptide have different amino acid sequences in thecorresponding portions.
 8. An isolated polynucleotide comprising anucleic acid molecule encoding a chimeric PIP-72 polypeptide comprisingat least a first moiety comprising a portion of a first PIP-72polypeptide encoded by the nucleic acid molecule of claim 1 and a secondmoiety comprising a complementary portion of a second PIP-72 polypeptideencoded by the nucleic acid molecule of claim 1, wherein the firstPIP-72 polypeptide and second PIP-72 polypeptide have different aminoacid sequences in the corresponding portions.
 9. An expression cassettecomprising the isolated polynucleotide of claim 8 operably linked to aheterologous regulatory element.
 10. The expression cassette of claim 9,wherein the regulatory element is a promoter capable of expressing aprotein in plants.
 11. A transgenic plant comprising the DNA constructof claim
 1. 12. A transgenic plant stably transformed with the DNAconstruct claim
 1. 13. A seed produced from the plant of claim 11 or 12,wherein the seed comprises the nucleic acid molecule.
 14. A progenyplant produced from the seed of claim
 13. 15. A host cell transformedwith the DNA construct of claim
 1. 16. The host cell of claim 15,wherein the host cell is a bacterial cell or a plant cell.
 17. The hostcell of claim 16, wherein the plant cell is a monocot or dicot plantcell.
 18. A recombinant PIP-72 polypeptide having insecticidal activityagainst Western corn rootworm (Diabrotica virgifera virgifera)comprising an amino acid sequence having at least 80% sequence identityto SEQ ID NO: 2 fused to a heterologous signal peptide or transitpeptide.
 19. The recombinant PIP-72 polypeptide of claim 18, wherein thePIP-72 polypeptide comprises 1 to 17 amino acid substitutions atposition 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 56, 57, 58,59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85 or 86, compared to the correspondingamino acid of SEQ ID NO:
 2. 20. The recombinant PIP-72 polypeptide ofclaim 18, wherein the PIP-72 polypeptide comprises 1 to 17 amino acidsubstitutions at position 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 17, 18, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, 40, 42, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54,56, 58, 60, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77,78, 79, 80, 81, 82, 83, 84, 85 or 86 compared to the corresponding aminoacid of SEQ ID NO:
 2. 21. The recombinant PIP-72 polypeptide of claim18, wherein the PIP-72 polypeptide comprises an amino acid sequence ofthe formula: SEQ ID NO: 846Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ala1               5                   10                  15Xaa Xaa Xaa Xaa Gly Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa            20                  25                  30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa        35                  40                  45Xaa Xaa Xaa Xaa Xaa Xaa Gly Xaa Gln Xaa Pro Xaa Tyr Val Xaa Xaa    50                  55                  60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65                  70                  75                  80Xaa Xaa Xaa Xaa Xaa Xaa                 85

wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu,Asn, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 3 is Ile or Trp;Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu, Arg, Ser,Val, Trp or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His, Ile orTyr; Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, Ile, Lys, Met,Pro, Gln, Arg, Ser, Trp or Tyr; Xaa at position 7 is Asn, Ala or Val;Xaa at position 8 is Asn, Ala, Cys, Asp, Glu, Gly, His, Ile, Lys, Leu,Met, Gln, Arg, Ser, Thr or Val; Xaa at position 9 is Ser, Ala, Cys, Glyor Thr; Xaa at position 10 is Ser, Ala, Glu, Phe, Gly, His, Ile, Lys,Leu, Asn, Pro, Gln, Arg, Thr or Trp; Xaa at position 11 is Asn, Ala,Cys, Asp, Glu, Gly, His, Ile, Lys, Leu, Met, Gln, Ser, Thr, Val or Tyr;Xaa at position 12 is Pro, Ala, Cys, Asp, Glu, Gly, His, Lys, Leu, Asn,Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 13 is Ile, Asn, Glnor Val; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys or Gln; Xaaat position 15 is Val, Ala, Cys, Ile, Met or Arg; Xaa at position 17 isIle, Glu or Val; Xaa at position 18 is Asn or Ser; Xaa at position 19 isHis, Ala, Glu, Lys, Leu, Pro, Arg, Ser or Tyr; Xaa at position 20 isTrp, Ala or Thr; Xaa at position 22 is Ser, Ala, Asp, Phe, Gly, His,Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr, Val or Tyr; Xaa at position23 is Asp, Ala, Gly, His, Lys, Met, Asn, Gln, Ser, Thr or Val; Xaa atposition 24 is Gly, Asp or Phe; Xaa at position 25 is Asp, Ala, Glu,Phe, Asn or Gln; Xaa at position 26 is Thr, Glu or Pro; Xaa at position27 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Asn, Gln, Arg or Thr; Xaaat position 28 is Phe, Pro, Trp or Tyr; Xaa at position 29 is Phe, Ala,Cys, Ile, Leu, Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Ala,Cys, Asp, Glu, Phe, Gly, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val, Trp or Tyr; Xaa at position 31 is Val, Ile or Leu; Xaa at position32 is Gly, Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg,Ser, Thr, Val, Trp or Tyr; Xaa at position 33 is Asn, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Lys, Leu, Pro, Gln, Arg, Ser, Thr, Val or Tyr;Xaa at position 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg,Ser, Thr or Tyr; Xaa at position 35 is Lys, Ala, Cys, Asp, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa at position 36 isGln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr orVal; Xaa at position 37 is Glu, Ala, Cys, Asp, Phe, Gly, Ile, Lys, Leu,Met, Asn, Ser, Thr or Val; Xaa at position 38 is Thr, Ala, Cys, Asp,Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Val, Trp or Tyr;Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; Xaa at position 42 is Ser, Ala, Cys, Asp, Glu, Phe, Gly,Ile, Lys, Leu, Met, Asn, Gln, Arg, Thr, Val, Trp or Tyr; Xaa at position44 is Ser, Ala, Asp, Glu, Gly, Leu, Met, Asn, Pro, Gln, Thr, Val or Tyr;Xaa at position 45 is Arg, Lys or Ser; Xaa at position 46 is Gly, Ala orGln; Xaa at position 47 is Phe, Cys, Val or Tyr; Xaa at position 48 isVal, Ile or Leu; Xaa at position 49 is Leu, Cys, Phe, Met, Arg or Tyr;Xaa at position 50 is Ser, Ala, Cys, Asp, Ile, Met, Pro, Gln, Thr orVal; Xaa at position 51 is Leu, Ala, Cys, Met or Val; Xaa at position 52is Lys, Cys, Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Trp or Tyr;Xaa at position 53 is Lys, Ala, Cys, Asp, Glu, Phe, His, Ile, Leu, Met,Asn, Gln, Arg, Ser, Thr, Val or Tyr; Xaa at position 54 is Asn, Cys,Asp, Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position 56is Ala, Gly, Leu, Asn, Pro, Gln, Arg, Ser or Thr; Xaa at position 57 isGln, Glu, Leu, Met, Ser or Thr; Xaa at position 58 is His, Ala, Asp,Phe, Leu, Met, Asn, Arg, Trp or Tyr; Xaa at position 60 is Tyr, Glu orPhe; Xaa at position 63 is Gln, Cys, Gly, Ile, Leu, Met, Asn, Thr, Valor Tyr; Xaa at position 64 is Ala, Phe, Gly, His, Arg, Ser or Tyr; Xaaat position 65 is Ser, Ala, Cys, Asp, Glu, Phe, Gly, His, Ile, Leu, Asn,Thr or Val; Xaa at position 66 is Ser, Ala or Gly; Xaa at position 67 isLys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu or Val; Xaa atposition 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Cys or Ile; Xaa atposition 71 is Asp, Ala, Cys, Gly, His, Ile, Leu, Met, Asn, Ser, Thr,Val or Tyr; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu, Gly, Lys,Met, Pro, Gln, Arg, Ser, Thr, Val or Trp; Xaa at position 73 is Asn,Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Ser, Thr, Val or Tyr; Xaa atposition 74 is Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 75 is Val, Cys, Ile or Leu; Xaa atposition 76 is Lys, Ala, Cys, Phe, His, Ile, Leu, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 77 is Asp Tyr; Xaa at position 78 isGln, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Met, Asn, Arg, Ser, Thr,Val or Tyr; Xaa at position 79 is Gly, Arg, Ala, Cys, Asp, Glu, Phe,His, Lys, Leu, Asn, Gln, Arg, Ser, Thr, Trp or Tyr; Xaa at position 80is Arg, Ala, Cys, Asp, Phe, Gly, His, Ile, Leu, Asn, Ser, Thr, Val orTyr; Xaa at position 81 is Leu, Ala, Cys, Asp, Phe, Gly, His, Ile, Asn,Pro, Arg, Ser, Thr or Val; Xaa at position 82 is Ile, Ala, Leu, Met, Argor Val; Xaa at position 83 is Glu, Ala, Cys, Asp, Phe, Gly, His, Ile,Lys, Leu, Asn, Pro, Arg, Ser, Thr, Val or Tyr; Xaa at position 84 isPro, Ala, Cys, Glu, Ile, Ser, Val, Trp or Tyr; Xaa at position 85 isLeu, Cys, Gly or Val; and Xaa at position 86 is Ser, Ala, Ile, Thr orVal.
 22. The recombinant PIP-72 polypeptide of claim 18, wherein thePIP-72 polypeptide comprises an amino acid sequence of the formula:SEQ ID NO: 849Met Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Ser Xaa Xaa Xaa Xaa Xaa Xaa1               5                   10                  15Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa            20                  25                  30Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Arg Xaa Asp Xaa Xaa Xaa Xaa Xaa        35                  40                  45Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Tyr Xaa Xaa Xaa    50                  55                  60Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa Xaa65                  70                  75                  80Xaa Xaa Xaa Xaa Xaa Xaa                 85

wherein Xaa at position 2 is Gly, Ala, Cys, Asp, Glu, Ile, Lys, Leu,Asn, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 3 is Ile, Leu, Valor Trp; Xaa at position 4 is Thr, Ala, Asp, Glu, His, Ile, Lys, Leu,Arg, Ser, Val, Trp or Tyr; Xaa at position 5 is Val, Ala, Cys, Gly, His,Ile, Leu or Tyr; Xaa at position 6 is Thr, Ala, Cys, Phe, Gly, His, Ile,Lys, Met, Pro, Gln, Arg, Ser, Trp or Tyr; Xaa at position 7 is Asn, Alaor Val; Xaa at position 8 is Asn, Lys, Gly, Ser, Gln, Arg, Thr, Ala,Cys, Asp, Glu, His, Ile, Leu, Met or Val; Xaa at position 9 is Ser, Ala,Cys, Gly or Thr; Xaa at position 11 is Asn, Lys, Thr, Gln, Arg, Ser,Ala, Cys, Asp, Glu, Gly, His, Ile, Leu, Met, Val or Tyr; Xaa at position12 is Pro, Thr, Lys, Ser, Arg, Ala, Cys, Asp, Glu, Gly, His, Leu, Asn,Gln, Arg, Val, Trp or Tyr; Xaa at position 13 is Ile, Asn, Gln, Leu orVal; Xaa at position 14 is Glu, Ala, Cys, Phe, His, Lys, Asp or Gln; Xaaat position 15 is Val, Ala, Ile, Leu, Cys, Met or Arg; Xaa at position16 is Ala or Ser; Xaa at position 17 is Ile, Glu, Leu or Val; Xaa atposition 18 is Asn, Gln, Thr or Ser; Xaa at position 19 is His, Lys,Ala, Arg, Glu, Leu, Pro, Ser or Tyr; Xaa at position 20 is Trp, Ala orThr; Xaa at position 21 is Gly, Arg or Lys; Xaa at position 22 is Ser,Ala, Asp, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Pro, Gln, Arg, Thr,Val or Tyr; Xaa at position 23 is Asp, Ala, Gly, His, Lys, Met, Asn,Gln, Ser, Thr or Val; Xaa at position 24 is Gly, Asp or Phe; Xaa atposition 25 is Asp, Ala, Glu, Phe, Asn or Gln; Xaa at position 26 isThr, Glu, Asp, Ser or Pro; Xaa at position 27 is Ser, Thr, Lys, Arg,Ala, Cys, Asp, Glu, Phe, Gly, His, Asn or Gln; Xaa at position 28 isPhe, Tyr, Pro or Trp; Xaa at position 29 is Phe, Ala, Cys, Ile, Leu,Gln, Arg, Trp or Tyr; Xaa at position 30 is Ser, Gly, Lys, Thr, Arg,Ala, Cys, Asp, Glu, Phe, His, Leu, Met, Asn, Pro, Gln, Val, Trp or Tyr;Xaa at position 31 is Val, Ile, Met or Leu; Xaa at position 32 is Gly,Ala, Asp, Glu, Phe, His, Lys, Leu, Met, Asn, Pro, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 33 is Asn, Ser, Gln, Pro, Thr, Ala,Cys, Asp, Glu, Phe, Gly, His, Ile, Lys, Leu, Arg, Val or Tyr; Xaa atposition 34 is Gly, Glu, Phe, His, Lys, Leu, Met, Asn, Gln, Arg, Ser,Thr or Tyr; Xaa at position 35 is Lys, Glu, Ala, Cys, Asp, Gly, His,Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr or Val; Xaa at position 36 isGln, Ala, Cys, Glu, Gly, His, Ile, Lys, Leu, Asn, Pro, Arg, Ser, Thr orVal; Xaa at position 37 is Glu, Asp, Ala, Cys, Phe, Gly, Ile, Lys, Leu,Met, Asn, Ser, Thr or Val; Xaa at position 38 is Thr, Ser, Ala, Cys,Asp, Glu, Phe, Gly, His, Ile, Leu, Met, Asn, Gln, Arg, Val, Trp or Tyr;Xaa at position 39 is Trp or Phe; Xaa at position 40 is Asp, Ala, Cys,Glu, Phe, Gly, His, Ile, Lys, Leu, Met, Asn, Gln, Arg, Ser, Thr, Val,Trp or Tyr; Xaa at position 42 is Ser, Asn, Thr, Ala, Cys, Asp, Glu,Phe, Gly, Ile, Lys, Leu, Met, Arg, Val, Trp, Tyr or Gln; Xaa at position44 is Ser, Asp, Ala, Leu, Thr, Glu, Ile, Ala, Gly, Leu, Met, Asn, Pro,Gln, Val, Tyr or Val; Xaa at position 45 is Arg, Lys or Ser; Xaa atposition 46 is Gly, Ala or Gln; Xaa at position 47 is Phe, Tyr Cys, Valor Trp; Xaa at position 48 is Leu, Met, Ile, Cys, Phe, Met, Arg, Tyr orVal; Xaa at position 49 is Leu, Met, Ile or Val; Xaa at position 50 isSer, Ala, Tyr, Cys, Asp, Ile, Met, Pro, Gln, Val or Thr; Xaa at position51 is Leu, Val, Ala, Cys, Met or Ile; Xaa at position 52 is Lys, Cys,Phe, His, Ile, Leu, Met, Asn, Arg, Ser, Thr, Gln, Trp or Tyr; Xaa atposition 53 is Lys, Arg, Met, Leu, Ile, Ala, Cys, Asp, Glu, Phe, His,Asn, Gln, Ser, Thr, Tyr or Val; Xaa at position 54 is Asn, Cys, Asp,Glu, Phe, Gly, Lys, Met, Gln, Arg, Ser or Trp; Xaa at position 55 isGly, Ser or Thr; Xaa at position 56 is Ala, Thr, Gln, Ser, Gly, Leu,Pro, Arg or Asn; Xaa at position 57 is Gln, Glu, Leu, Met, Ser, Val,Ala, Asn, Ile or Thr; Xaa at position 58 is His, Ala, Lys, Asp, Phe,Leu, Met, Asn, Arg, Trp, Tyr or Thr; Xaa at position 59 is Pro, Thr orSer; Xaa at position 60 is Tyr, Glu or Phe; Xaa at position 62 is Val,Ile or Leu; Xaa at position 63 is Gln, Ser, Cys, Gly, Ile, Leu, Met,Asn, Thr, Val or Tyr; Xaa at position 64 is Ala, Gln, Asn, Phe, Gly,His, Arg, Ser or Tyr; Xaa at position 65 is Ser, Ala, Cys, Asp, Glu,Phe, Gly, His, Ile, Leu, Asn, Val or Thr; Xaa at position 66 is Ser, Alaor Gly; Xaa at position 67 is Lys, Gln, Asn or Arg; Xaa at position 67is Lys, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Asn, Gln, Arg, Ser, Thr,Val, Trp or Tyr; Xaa at position 68 is Ile Asp, Leu or Val; Xaa atposition 69 is Glu, Ala, Cys, Asp, Phe, His, Ile, Leu, Met, Gln, Arg,Ser, Thr, Val or Tyr; Xaa at position 70 is Val, Ile, Cys or Leu; Xaa atposition 71 is Asp, Glu, Tyr, Ala, Cys, Gly, His, Ile, Leu, Met, Asn,Ser, Thr, Val or Trp; Xaa at position 72 is Asn, Ala, Cys, Asp, Glu,Gly, Lys, Met, Pro, Gln, Arg, Ser, Thr, Val, His or Trp; Xaa at position73 is Asn, Ser, Asp, Gln, Thr, Ala, Cys, Phe, Gly, His, Ile, Leu, Val,Tyr or Glu; Xaa at position 74 is Ala, Thr, Met, Ile, Lys, Ser, Leu,Val, Cys, Asp, Phe, Gly, His, Asn, Gln, Tyr or Arg; Xaa at position 75is Val, Cys, Ile or Leu; Xaa at position 76 is Lys, Ala, Cys, Phe, His,Ile, Leu, Gln, Arg, Ser, Thr, Val, Trp or Tyr; Xaa at position 77 is AspTyr; Xaa at position 78 is Gln, His, Ser, Asn, Ala, Cys, Asp, Phe, Gly,Ile, Leu, Met, Asn, Arg, Val, Tyr or Thr; Xaa at position 79 is Gly,Arg, Ala, Cys, Asp, Glu, Phe, His, Lys, Leu, Asn, Gln, Arg, Ser, Thr,Trp or Tyr; Xaa at position 80 is Arg, Glu, Gln, Lys, Asp, Ala, Cys,Phe, Gly, His, Ile, Leu, Ser, Thr, Val, Tyr or Asn; Xaa at position 81is Leu, Pro, Thr, Ile, Val, Ala, Cys, Asp, Phe, Gly, His or Ser; Xaa atposition 82 is Ile, Ala, Leu, Met, Arg and Val; Xaa at position 83 isGlu, His, Asn, Leu, Gln, Ile, Ala, Cys, Asp, Phe, Gly, Lys, Pro, Arg,Ser, Thr, Tyr or Val; Xaa at position 84 is Pro, Ala, Cys, Glu, Ile,Ser, Val, Trp or Tyr; Xaa at position 85 is Leu, Val, Cys, Gly or Ala;and Xaa at position 86 is Ser, Ala, Tyr, Asn, Ile, Val or Thr.
 23. Therecombinant PIP-72 polypeptide of claim 18, wherein the PIP-72polypeptide comprises an amino acid motif as represented by positions37-51 of SEQ ID NO: 846, SEQ ID NO: 847, SEQ ID NO: 848 or SEQ ID NO:849.
 24. A chimeric PIP-72 polypeptide comprising at least a firstmoiety comprising a portion of a first PIP-72 polypeptide of claim 18and a second moiety comprising a complementary portion of a secondPIP-72 polypeptide of claim 18, wherein the first PIP-72 polypeptide andsecond PIP-72 polypeptide have different amino acid sequences in thecorresponding portions.
 25. A composition comprising the recombinantPIP-72 polypeptide of claim
 18. 26. A fusion protein comprising thePIP-72 polypeptide of claim
 18. 27. A method for controlling cornrootworm, comprising contacting an insect pest population with aninsecticidally-effective amount of the PIP-72 polypeptide of claim 18.28. A method of inhibiting growth or killing corn rootworm, comprisingcontacting the insect with a composition comprising aninsecticidally-effective amount of the PIP-72 polypeptide of claim 18.29. A method for controlling a corn rootworm population resistant to apesticidal protein, comprising contacting the insect population with aninsecticidally-effective amount of the PIP-72 polypeptide of claim 18.30. A method of controlling a corn rootworm infestation in a transgenicplant and providing insect resistance management, comprising expressingin the plant the PIP-72 polypeptide of claim 18.